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Sample records for alcohol dehydrogenases adhs

  1. 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

  2. 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.

  3. [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.

  4. 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

  5. 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.

  6. 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

  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. 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

  9. 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%.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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

  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. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  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. 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.

  8. 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

  9. Rare ADH Variant Constellations are Specific for Alcohol Dependence

    PubMed Central

    Zuo, Lingjun; Zhang, Heping; Malison, Robert T.; Li, Chiang-Shan R.; Zhang, Xiang-Yang; Wang, Fei; Lu, Lingeng; Lu, Lin; Wang, Xiaoping; Krystal, John H.; Zhang, Fengyu; Deng, Hong-Wen; Luo, Xingguang

    2013-01-01

    Aims: Some of the well-known functional alcohol dehydrogenase (ADH) gene variants (e.g. ADH1B*2, ADH1B*3 and ADH1C*2) that significantly affect the risk of alcohol dependence are rare variants in most populations. In the present study, we comprehensively examined the associations between rare ADH variants [minor allele frequency (MAF) <0.05] and alcohol dependence, with several other neuropsychiatric and neurological disorders as reference. Methods: A total of 49,358 subjects in 22 independent cohorts with 11 different neuropsychiatric and neurological disorders were analyzed, including 3 cohorts with alcohol dependence. The entire ADH gene cluster (ADH7–ADH1C–ADH1B–ADH1A–ADH6–ADH4–ADH5 at Chr4) was imputed in all samples using the same reference panels that included whole-genome sequencing data. We stringently cleaned the phenotype and genotype data to obtain a total of 870 single nucleotide polymorphisms with 0< MAF <0.05 for association analysis. Results: We found that a rare variant constellation across the entire ADH gene cluster was significantly associated with alcohol dependence in European-Americans (Fp1: simulated global P = 0.045), European-Australians (Fp5: global P = 0.027; collapsing: P = 0.038) and African-Americans (Fp5: global P = 0.050; collapsing: P = 0.038), but not with any other neuropsychiatric disease. Association signals in this region came principally from ADH6, ADH7, ADH1B and ADH1C. In particular, a rare ADH6 variant constellation showed a replicable association with alcohol dependence across these three independent cohorts. No individual rare variants were statistically significantly associated with any disease examined after group- and region-wide correction for multiple comparisons. Conclusion: We conclude that rare ADH variants are specific for alcohol dependence. The ADH gene cluster may harbor a causal variant(s) for alcohol dependence. PMID:23019235

  10. 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.

  11. 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

  12. In vivo roles of alcohol dehydrogenase (ADH), catalase and the microsomal ethanol oxidizing system (MEOS) in deermice

    SciTech Connect

    Takagi, T.; Alderman, J.; Lieber, C.S.

    1985-01-01

    The relative importance of ADH and MEOS for ethanol oxidation in the liver has yet to be elucidated. The discovery of a strain of deermice genetically lacking ADH (ADH-) which can consume ethanol at greater than 50% of the rates seen in deermice having ADH (ADH+) suggested a significant role for non-ADH pathways in vivo. To quantitate contributions of the various pathways, the authors examined first the ethanol oxidation rates with or without 4-methylpyrazole in isolated deermice hepatocytes. 4-Methylpyrazole significantly reduced the ethanol oxidation in both ADH+ and ADH- hepatocytes. The reduction seen in ADH- cells can be applied to correct for the effect of 4-methylpyrazole on non-ADH pathways of ADH+ deermouse hepatocytes. After correction, non-ADH pathways were found to contribute 28% of ethanol metabolism at 10 mM and 52% at 50 mM. When using a different approach namely measurement of the isotope effect, MEOS was calculated to account for 35% at low and about 70% at high blood ethanol concentrations. Thus, they found that two different complementary approaches yielded similar results, namely that non-ADH pathways play a significant role in ethanol oxidation even in the presence of ADH.

  13. Genetic polymorphisms of ADH1B, ADH1C and ALDH2 in Turkish alcoholics: lack of association with alcoholism and alcoholic cirrhosis.

    PubMed

    Vatansever, Sezgin; Tekin, Fatih; Salman, Esin; Altintoprak, Ender; Coskunol, Hakan; Akarca, Ulus Salih

    2015-05-17

    No data exists regarding the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) gene polymorphisms in Turkish alcoholic cirrhotics. We studied the polymorphisms of ADH1B, ADH1C and ALDH2 genes in alcoholic cirrhotics and compared the results with non-cirrhotic alcoholics and healthy volunteers. Overall, 237 subjects were included for the study: 156 alcoholic patients (78 cirrhotics, 78 non-cirrhotic alcoholics) and 81 healthy volunteers. Three different single-nucleotide-polymorphism genotyping methods were used. ADH1C genotyping was performed using a polymerase chain reaction-restriction fragment length polymorphism method. The identified ADH1C genotypes were named according to the presence or absence of the enzyme restriction sites. ADH1B (Arg47Hys) genotyping was performed using the allele specific primer extension method, and ALDH2 (Glu487Lys) genotyping was performed by a multiplex polymerase chain reaction using two allele-specific primer pairs. For ADH1B, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 97.4%, 94.9% and 99.4%, respectively. For ADH1C, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 47%, 36.3% and 45%, respectively. There was no statistical difference between the groups for ADH1B and ADH1C (p>0.05). All alcoholic and non-alcoholic subjects (100%) had the allele *1 for ALDH2. The obtained results for ADH1B, ADH1C, and ALDH gene polymorphisms in the present study are similar to the results of Caucasian studies. ADH1B and ADH1C genetic variations are not related to the development of alcoholism or susceptibility to alcoholic cirrhosis. ALDH2 gene has no genetic variation in the Turkish population.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. ADH2 gene polymorphisms are determinants of alcohol pharmacokinetics.

    PubMed

    Thomasson, H R; Beard, J D; Li, T K

    1995-12-01

    The class I hepatic alcohol dehydrogenases (ADHs) are primarily responsible for ethanol metabolism in humans. Genetic polymorphism at the ADH2 locus results in the inheritance of isozymes of strikingly different catalytic properties. The most common ADH2 allele, ADH2*1, encodes the low K(m) isozyme subunit beta 1. The ADH2*3 allele encodes a high-activity isozyme subunit of alcohol dehydrogenase, beta 3, identified in approximately 25% of African-Americans. The Vmax of beta 3 beta 3-ADH is 30 times greater than that of the beta 1 beta 1-ADH. Therefore, we hypothesized that the rate of ethanol metabolism, an important factor in the toxicity of ethanol, in persons with beta 3-containing ADH, either beta 3 beta 3- or beta 1 beta 3-ADH, would be faster than that of persons with only beta 1 beta 1-ADH. We tested this hypothesis with ethanol administered orally to healthy, young African-Americans. Three hundred and twenty-six African-American men and women were genotyped using polymerase chain reaction amplification of their leukocyte DNA followed by hybridization with allele-specific probes. One hundred twelve volunteers, selected by genotype, received an oral dose of ethanol designed to produce a blood ethanol concentration of 80 mg/dl (0.080 g/dl), when the blood alcohol concentration-time curve was extrapolated back to time 0. Ethanol metabolic rates (beta 60s) were determined in the 112 subjects from the slope of the pseudolinear portion of the blood ethanol concentration-time curves. The mean beta 60 of African-Americans having beta 3-containing ADH isozymes had significantly faster ethanol elimination rates than those with only beta 1 beta 1-ADH isozymes. There were no significant differences in body weight, ethanol intake in the week before testing, peak breath ethanol concentration, time to peak, or volume of distribution between the genotype groups. Within each of these groups, men had lower ethanol disappearance rates than women. These results demonstrate in

  19. Allelic variants of ADH, ALDH and the five factor model of personality in alcohol dependence syndrome

    PubMed Central

    Salujha, S. K.; Chaudhury, S.; Menon, P. K.; Srivastava, K.; Gupta, A.

    2014-01-01

    Background: The etiology of alcohol dependence is a complex interplay of biopsychosocial factors. The genes for alcohol-metabolizing enzymes: Alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) exhibit functional polymorphisms. Vulnerability of alcohol dependence may also be in part due to heritable personality traits. Aim: To determine whether any association exists between polymorphisms of ADH2, ADH3 and ALDH2 and alcohol dependence syndrome in a group of Asian Indians. In addition, the personality of these patients was assessed to identify traits predisposing to alcoholism. Materials and Methods: In this study, 100 consecutive males with alcohol dependence syndrome attending the psychiatric outpatient department of a tertiary care service hospital and an equal number of matched healthy controls were included with their consent. Blood samples of all the study cases and controls were collected and genotyped for the ADH2, ADH3 and ALDH2 loci. Personality was evaluated using the neuroticism, extraversion, openness (NEO) personality inventory and sensation seeking scale. Results: Allele frequencies of ADH2*2 (0.50), ADH3*1 (0.67) and ALSH2*2 (0.09) were significantly low in the alcohol dependent subjects. Personality traits of NEO personality inventory and sensation seeking were significantly higher when compared to controls. Conclusions: The functional polymorphisms of genes coding for alcohol metabolizing enzymes and personality traits of NEO and sensation seeking may affect the propensity to develop dependence. PMID:25535445

  20. 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.

  1. 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.

  2. 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

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

    PubMed Central

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

    2016-01-01

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

  4. 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

  5. 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.

  6. ADH single nucleotide polymorphism associations with alcohol metabolism in vivo

    PubMed Central

    Birley, Andrew J.; James, Michael R.; Dickson, Peter A.; Montgomery, Grant W.; Heath, Andrew C.; Martin, Nicholas G.; Whitfield, John B.

    2009-01-01

    We have previously found that variation in alcohol metabolism in Europeans is linked to the chromosome 4q region containing the ADH gene family. We have now typed 103 single nucleotide polymorphisms (SNPs) across this region to test for allelic associations with variation in blood and breath alcohol concentrations after an alcohol challenge. In vivo alcohol metabolism was modelled with three parameters that identified the absorption and rise of alcohol concentration following ingestion, and the rate of elimination. Alleles of ADH7 SNPs were associated with the early stages of alcohol metabolism, with additional effects in the ADH1A, ADH1B and ADH4 regions. Rate of elimination was associated with SNPs in the intragenic region between ADH7 and ADH1C, and across ADH1C and ADH1B. SNPs affecting alcohol metabolism did not correspond to those reported to affect alcohol dependence or alcohol-related disease. The combined SNP associations with early- and late-stage metabolism only account for approximately 20% of the total genetic variance linked to the ADH region, and most of the variance for in vivo alcohol metabolism linked to this region is yet to be explained. PMID:19193628

  7. ADH and ALDH polymorphisms and alcohol dependence in Mexican and Native Americans

    PubMed Central

    Ehlers, Cindy L.; Liang, Tiebing; Gizer, Ian R.

    2012-01-01

    Background Ethanol is primarily metabolized in the liver by 2 rate-limiting reactions: conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) and subsequent conversion of acetaldehyde to acetate by aldehyde dehydrogenase (ALDH). ADH and ALDH exist in multiple isozymes that differ in their kinetic properties. Notably, polymorphisms within the genes that encode for these isozymes vary in their allele frequencies between ethnic groups, and thus, they have been considered as candidate genes that may differentially influence risk for the development of alcohol dependence across ethnic groups. Objectives and Methods Associations between alcohol dependence and polymorphisms in ADH1B, ADH1C, and ALDH2, were compared in a community sample of Native Americans living on reservations (n=791) and Mexican Americans (n=391) living within the same county. Results Two Mexican Americans and no Native Americans possessed one ALDH2*2 allele. Presence of at least one ADH1B*2 allele was found in 7% of the Native Americans and 13% of the Mexican Americans, but was only associated with protection against alcohol dependence in the Mexican Americans. Presence of at least one ADH1B*3 allele was found in 4% if the Native Americans and 2% of the Mexican Americans, but was associated with protection against alcohol dependence only in the Native Americans. No associations between alcohol dependence and polymorphisms in ADH1C were found. Conclusions and Scientific Significance Polymorphisms in ADH1B are protective against alcoholism in these two populations; however, these findings do not explain the high prevalence of alcoholism in these populations. PMID:22931071

  8. ADH and ALDH polymorphisms and alcohol dependence in Mexican and Native Americans

    PubMed Central

    Ehlers, Cindy L.; Liang, Tiebing; Gizer, Ian R.

    2012-01-01

    Background Ethanol is primarily metabolized in the liver by 2 rate-limiting reactions: conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) and subsequent conversion of acetaldehyde to acetate by aldehyde dehydrogenase (ALDH). ADH and ALDH exist in multiple isozymes that differ in their kinetic properties. Notably, polymorphisms within the genes that encode for these isozymes vary in their allele frequencies between ethnic groups, and thus, they have been considered as candidate genes that may differentially influence risk for the development of alcohol dependence across ethnic groups. Objectives and Methods Associations between alcohol dependence and polymorphisms in ADH1B, ADH1C, and ALDH2, were compared in a community sample of Native Americans living on reservations (n=791) and Mexican Americans (n=391) living within the same county. Results Two Mexican Americans and no Native Americans possessed one ALDH2*2 allele. Presence of at least one ADH1B*2 allele was found in 7% of the Native Americans and 13% of the Mexican Americans, but was only associated with protection against alcohol dependence in the Mexican Americans. Presence of at least one ADH1B*3 allele was found in 4% if the Native Americans and 2% of the Mexican Americans, but was associated with protection against alcohol dependence only in the Native Americans. No associations between alcohol dependence and polymorphisms in ADH1C were found. Conclusions and Scientific Significance Polymorphisms in ADH1B are protective against alcoholism in these two populations; however, these findings do not explain the high prevalence of alcoholism in these populations. PMID:22931071

  9. 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

  10. Polymorphisms in Alcohol Metabolism Genes ADH1B and ALDH2, Alcohol Consumption and Colorectal Cancer

    PubMed Central

    Crous-Bou, Marta; Rennert, Gad; Cuadras, Daniel; Salazar, Ramon; Cordero, David; Saltz Rennert, Hedy; Lejbkowicz, Flavio; Kopelovich, Levy; Monroe Lipkin, Steven; Bernard Gruber, Stephen; Moreno, Victor

    2013-01-01

    Background Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included alcohol intake, which is mainly metabolized to acetaldehyde by alcohol dehydrogenase and further oxidized to acetate by aldehyde dehydrogenase; consequently, the role of genes in the alcohol metabolism pathways is of particular interest. The aim of this study is to analyze the association between SNPs in ADH1B and ALDH2 genes and CRC risk, and also the main effect of alcohol consumption on CRC risk in the study population. Methodology/Principal Findings SNPs from ADH1B and ALDH2 genes, included in alcohol metabolism pathway, were genotyped in 1694 CRC cases and 1851 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. A positive association between alcohol consumption and CRC risk was observed in male participants from the Molecular Epidemiology of Colorectal Cancer study (MECC) study (OR = 1.47; 95%CI = 1.18-1.81). Moreover, the SNPs rs1229984 in ADH1B gene was found to be associated with CRC risk: under the recessive model, the OR was 1.75 for A/A genotype (95%CI = 1.21-2.52; p-value = 0.0025). A path analysis based on structural equation modeling showed a direct effect of ADH1B gene polymorphisms on colorectal carcinogenesis and also an indirect effect mediated through alcohol consumption. Conclusions/Significance Genetic polymorphisms in the alcohol metabolism pathways have a potential role in colorectal carcinogenesis, probably due to the differences in the ethanol metabolism and acetaldehyde oxidation of these enzyme variants. PMID:24282520

  11. 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

  12. 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.

  13. Association of ADH and ALDH Genes With Alcohol Dependence in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) Sample

    PubMed Central

    Kuo, Po-Hsiu; Kalsi, Gursharan; Prescott, Carol A.; Hodgkinson, Colin A.; Goldman, David; van den Oord, Edwin J.; Alexander, Jeffry; Jiang, Cizhong; Sullivan, Patrick F.; Patterson, Diana G.; Walsh, Dermot; Kendler, Kenneth S.; Riley, Brien P.

    2008-01-01

    Background: The genes coding for ethanol metabolism enzymes [alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)] have been widely studied for their influence on the risk to develop alcohol dependence (AD). However, the relation between polymorphisms of these metabolism genes and AD in Caucasian subjects has not been clearly established. The present study examined evidence for the association of alcohol metabolism genes with AD in the Irish Affected Sib Pair Study of alcohol dependence. Methods: We conducted a case–control association study with 575 independent subjects who met Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, AD diagnosis and 530 controls. A total of 77 single nucleotide polymorphisms (SNPs) in the seven ADH (ADH1-7) and two ALDH genes (ALDH1A1 and ALDH2) were genotyped using the Illumina GoldenGate protocols. Several statistical procedures were implemented to control for false discoveries. Results: All markers with minor allele frequency greater than 0.01 were in Hardy–Weinberg equilibrium. Numerous SNPs in ADH genes showed association with AD, including one marker in the coding region of ADH1C (rs1693482 in exon6, Ile271Gln). Haplotypic association was observed in the ADH5 and ADH1C genes, and in a long haplotype block formed by the ADH1A and ADH1B loci. We detected two significant interactions between pairs of markers in intron 6 of ADH6 and intron 12 of ALDH2 (p = 5 × 10−5), and 5′ of both ADH4 and ADH1A (p = 2 × 10−4). Conclusion: We found evidence for the association of several ADH genes with AD in a sample of Western European origin. The significant interaction effects between markers in ADH and ALDH genes suggest possible epistatic roles between alcohol metabolic enzymes in the risk for AD. PMID:18331377

  14. 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.

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

    PubMed Central

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

    1997-01-01

    This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

  16. Genetic variants in or near ADH1B and ADH1C affect susceptibility to alcohol dependence in a British and Irish population.

    PubMed

    Way, Michael; McQuillin, Andrew; Saini, Jit; Ruparelia, Kush; Lydall, Gregory J; Guerrini, Irene; Ball, David; Smith, Iain; Quadri, Giorgia; Thomson, Allan D; Kasiakogia-Worlley, Katherine; Cherian, Raquin; Gunwardena, Priyanthi; Rao, Harish; Kottalgi, Girija; Patel, Shamir; Hillman, Audrey; Douglas, Ewen; Qureshi, Sherhzad Y; Reynolds, Gerry; Jauhar, Sameer; O'Kane, Aideen; Dedman, Alex; Sharp, Sally; Kandaswamy, Radhika; Dar, Karim; Curtis, David; Morgan, Marsha Y; Gurling, Hugh M D

    2015-05-01

    Certain single nucleotide polymorphisms (SNPs) in genes encoding alcohol dehydrogenase (ADH) enzymes confer a significant protective effect against alcohol dependence syndrome (ADS) in East Asian populations. Recently, attention has focused on the role of these SNPs in determining ADS risk in European populations. To further elucidate these associations, SNPs of interest in ADH1B, ADH1C and the ADH1B/1C intergenic region were genotyped in a British and Irish population (ADS cases n = 1076: controls n = 1027) to assess their relative contribution to ADS risk. A highly significant, protective association was observed between the minor allele of rs1229984 in ADH1B and ADS risk [allelic P = 8.4 × 10(-6) , odds ratio (OR) = 0.26, 95 percent confidence interval, 0.14, 0.49]. Significant associations were also observed between ADS risk and the ADH1B/1C intergenic variant, rs1789891 [allelic P = 7.2 × 10(-5) , OR = 1.4 (1.2, 1.6)] and three non-synonymous SNPs rs698, rs1693482 and rs283413 in ADH1C. However, these associations were not completely independent; thus, while the ADH1B rs1229984 minor allele association was independent of those of the intergenic variant rs1789891 and the three ADH1C variants, the three ADH1C variants were not individually independent. In conclusion, the rare ADH1B rs1229984 mutation provides significant protection against ADS in this British and Irish population; other variants in the ADH gene cluster also alter ADS risk, although the strong linkage disequilibrium between SNPs at this location precluded clear identification of the variant(s) driving the associations.

  17. 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

  18. 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.

  19. 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

  20. Diplotype Trend Regression Analysis of the ADH Gene Cluster and the ALDH2 Gene: Multiple Significant Associations with Alcohol Dependence

    PubMed Central

    Luo, Xingguang; Kranzler, Henry R.; Zuo, Lingjun; Wang, Shuang; Schork, Nicholas J.; Gelernter, Joel

    2006-01-01

    The set of alcohol-metabolizing enzymes has considerable genetic and functional complexity. The relationships between some alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes and alcohol dependence (AD) have long been studied in many populations, but not comprehensively. In the present study, we genotyped 16 markers within the ADH gene cluster (including the ADH1A, ADH1B, ADH1C, ADH5, ADH6, and ADH7 genes), 4 markers within the ALDH2 gene, and 38 unlinked ancestry-informative markers in a case-control sample of 801 individuals. Associations between markers and disease were analyzed by a Hardy-Weinberg equilibrium (HWE) test, a conventional case-control comparison, a structured association analysis, and a novel diplotype trend regression (DTR) analysis. Finally, the disease alleles were fine mapped by a Hardy-Weinberg disequilibrium (HWD) measure (J). All markers were found to be in HWE in controls, but some markers showed HWD in cases. Genotypes of many markers were associated with AD. DTR analysis showed that ADH5 genotypes and diplotypes of ADH1A, ADH1B, ADH7, and ALDH2 were associated with AD in European Americans and/or African Americans. The risk-influencing alleles were fine mapped from among the markers studied and were found to coincide with some well-known functional variants. We demonstrated that DTR was more powerful than many other conventional association methods. We also found that several ADH genes and the ALDH2 gene were susceptibility loci for AD, and the associations were best explained by several independent risk genes. PMID:16685648

  1. The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr.

    PubMed Central

    Carlini, D B; Chen, Y; Stephan, W

    2001-01-01

    To gain insights into the relationship between codon bias, mRNA secondary structure, third-codon position nucleotide distribution, and gene expression, we predicted secondary structures in two related drosophilid genes, Adh and Adhr, which differ in degree of codon bias and level of gene expression. Individual structural elements (helices) were inferred using the comparative method. For each gene, four types of randomization simulations were performed to maintain/remove codon bias and/or to maintain or alter third-codon position nucleotide composition (N3). In the weakly expressed, weakly biased gene Adhr, the potential for secondary structure formation was found to be much stronger than in the highly expressed, highly biased gene Adh. This is consistent with the observation of approximately equal G and C percentages in Adhr ( approximately 31% across species), whereas in Adh the N3 distribution is shifted toward C (42% across species). Perturbing the N3 distribution to approximately equal amounts of A, G, C, and T increases the potential for secondary structure formation in Adh, but decreases it in Adhr. On the other hand, simulations that reduce codon bias without changing N3 content indicate that codon bias per se has only a weak effect on the formation of secondary structures. These results suggest that, for these two drosophilid genes, secondary structure is a relatively independent, negative regulator of gene expression. Whereas the degree of codon bias is positively correlated with level of gene expression, strong individual secondary structural elements may be selected for to retard mRNA translation and to decrease gene expression. PMID:11606539

  2. Fetal ADH2*3, maternal alcohol consumption, and fetal growth.

    PubMed

    Arfsten, Darryl P; Silbergeld, Ellen K; Loffredo, Christopher A

    2004-01-01

    There is some evidence suggesting the allele for alcohol dehydrogenase 2*3 (ADH2*3) is associated with a protective effect against alcohol-related intrauterine growth retardation (IUGR). This study was conducted to explore the affect of the ADH2*3 allele on fetal growth. Bloodspots (n = 1016) belonging to individual infants of a subgroup of the Baltimore-Washington Infant Study (BWIS) were assayed for the presence of the ADH2*3 allele by a polymerase chain reaction (PCR)-based method. Infants genotyped for ADH2*3 were those for whom bloodspots were identified and obtained from the Maryland Newborn Screening Program. The effect of ADH2*3 and maternal alcohol consumption on intrauterine growth was explored by multivariable linear regression analysis. Twenty-six percent of the 306 blood spots belonging to African-American infants were positive for ADH2*3 (4% were homozygous and 22% were heterozygous). Only a small percentage of bloodspots for Caucasian (1.3%) were positive for the ADH2*3 allele. Consequently, further analysis concentrated on gene-exposure interactions for African-American infants. It was found that the incidence of being small-for-gestation-age (SGA) was lower for ADH2*3-positive infants (2.5% versus 8.8%; p = .08). SGA infants had elevated odds for being ADH2*3 negative (OR: 3.15, 95% C.I.: 0.70-14.26) and for being born to mothers that consumed alcohol during pregnancy (OR: 2.31, 95% C.I.: 0.77-6.91). A negative trend between maternal alcohol consumption and mean offspring birthweight was found; however, ADH2*3 did not have a significant impact on mean birthweight for infants born to mothers that drank during pregnancy. These results could be interpreted as possible support for the hypothesis that ADH2 genotype in the infant may impact risk for alcohol-related IUGR. However, this study has limitations in that it is a "nested study of convenience" and involves a relatively small number of infants born to mothers reporting moderate to heavy alcohol

  3. 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.

  4. 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

  5. Polymorphism of ADH and ALDH genes among four ethnic groups in China and effects upon the risk for alcoholism.

    PubMed

    Shen, Y C; Fan, J H; Edenberg, H J; Li, T K; Cui, Y H; Wang, Y F; Tian, C H; Zhou, C F; Zhou, R L; Wang, J; Zhao, Z L; Xia, G Y

    1997-10-01

    The alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) that metabolize ethanol are polymorphic. Different alleles encode subunits of the enzymes that differ in their rate of metabolizing ethanol. These polymorphisms are distributed differently among populations and have been shown to influence the risk for alcoholism in some Asian populations. We have examined the allele frequencies at the ADH2, ADH3, and ALDH2 loci in four populations from China (Han, Mongolian, Korean, and Elunchun) and in alcoholics within each population. The four populations differ in allele frequencies, with the Elunchun having a much lower frequency of ADH2*2 alleles, and the Mongolian and Elunchun having a much lower frequency of ALDH2*2 alleles. Within each population, alleles at one or more of these three loci are protective against alcoholism, although the populations differ in which loci play significant roles. The protective allele at each locus (ALDH2*2, ADH2*2, and ADH3*1) encodes a subunit that either metabolizes ethanol to acetaldehyde more rapidly or slows the conversion of acetaldehyde to acetate. Taken as a whole, data demonstrate that genetic differences in the enzymes that metabolize alcohol can substantially affect the risk for alcoholism.

  6. 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

  7. Association and ancestry analysis of sequence variants in ADH and ALDH using alcohol-related phenotypes in a Native American community sample

    PubMed Central

    Peng, Qian; Gizer, Ian R.; Libiger, Ondrej; Bizon, Chris; Wilhelmsen, Kirk C.; Schork, Nicholas J.; Ehlers, Cindy L.

    2015-01-01

    Higher rates of alcohol use and other drug-dependence have been observed in some Native American populations relative to other ethnic groups in the U.S. Previous studies have shown that alcohol dehydrogenase (ADH) genes and aldehyde dehydrogenase (ALDH) genes may affect the risk of development of alcohol dependence, and that polymorphisms within these genes may differentially affect risk for the disorder depending on the ethnic group evaluated. We evaluated variations in the ADH and ALDH genes in a large study investigating risk factors for substance use in a Native American population. We assessed ancestry admixture and tested for associations between alcohol-related phenotypes in the genomic regions around the ADH1-7 and ALDH2 and ALDH1A1 genes. Seventy-two (72) ADH variants showed significant evidence of association with a severity level of alcohol drinking-related dependence symptoms phenotype. These significant variants spanned across the entire 7 ADH gene cluster regions. Two significant associations, one in ADH and one in ALDH2, were observed with alcohol dependence diagnosis. Seventeen (17) variants showed significant association with the largest number of alcohol drinks ingested during any 24-hour period. Variants in or near ADH7 were significantly negatively associated with alcohol-related phenotypes, suggesting a potential protective effect of this gene. In addition, our results suggested that a higher degree of Native American ancestry is associated with higher frequencies of potential risk variants and lower frequencies of potential protective variants for alcohol dependence phenotypes. PMID:25270064

  8. Association and ancestry analysis of sequence variants in ADH and ALDH using alcohol-related phenotypes in a Native American community sample.

    PubMed

    Peng, Qian; Gizer, Ian R; Libiger, Ondrej; Bizon, Chris; Wilhelmsen, Kirk C; Schork, Nicholas J; Ehlers, Cindy L

    2014-12-01

    Higher rates of alcohol use and other drug-dependence have been observed in some Native American (NA) populations relative to other ethnic groups in the US. Previous studies have shown that alcohol dehydrogenase (ADH) genes and aldehyde dehydrogenase (ALDH) genes may affect the risk of development of alcohol dependence, and that polymorphisms within these genes may differentially affect risk for the disorder depending on the ethnic group evaluated. We evaluated variations in the ADH and ALDH genes in a large study investigating risk factors for substance use in a NA population. We assessed ancestry admixture and tested for associations between alcohol-related phenotypes in the genomic regions around the ADH1-7 and ALDH2 and ALDH1A1 genes. Seventy-two ADH variants showed significant evidence of association with a severity level of alcohol drinking-related dependence symptoms phenotype. These significant variants spanned across the entire 7 ADH gene cluster regions. Two significant associations, one in ADH and one in ALDH2, were observed with alcohol dependence diagnosis. Seventeen variants showed significant association with the largest number of alcohol drinks ingested during any 24-hour period. Variants in or near ADH7 were significantly negatively associated with alcohol-related phenotypes, suggesting a potential protective effect of this gene. In addition, our results suggested that a higher degree of NA ancestry is associated with higher frequencies of potential risk variants and lower frequencies of potential protective variants for alcohol dependence phenotypes.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. Further clarification of the contribution of the ADH1C gene to vulnerability of alcoholism and selected liver diseases.

    PubMed

    Li, Dawei; Zhao, Hongyu; Gelernter, Joel

    2012-08-01

    The alcohol dehydrogenase 1C (ADH1C) subunit is an important member of the alcohol dehydrogenase family, a set of genes that plays a major role in the catabolism of ethanol. Numerous association studies have provided compelling evidence that ADH1C gene variation (formerly ADH3) is associated with altered genetic susceptibility to alcoholism and alcohol-related liver disease, cirrhosis, or pancreatitis. However, the results have been inconsistent, partially, because each study involved a limited number of subjects, and some were underpowered. Using cumulative data over the past two decades, this meta-analysis (6,796 cases and 6,938 controls) considered samples of Asian, European, African, and Native American origins to examine whether the aggregate genotype provide statistically significant evidence of association. The results showed strong evidence of association between ADH1C Ile350Val (rs698, formerly ADH1C *1/*2) and alcohol dependence (AD) and abuse in the combined studies. The overall allelic (Val vs. Ile or *2 vs. *1) P value was 1 × 10(-8) and odds ratio (OR) was 1.51 (1.31, 1.73). The Asian populations produced stronger evidence of association with an allelic P value of 4 × 10(-33) [OR 2.14 (1.89, 2.43)] with no evidence of heterogeneity, and the dominant and recessive models revealed even stronger effect sizes. The strong evidence remained when stricter criteria and sub-group analyses were applied, while Asians always showed stronger associations than other populations. Our findings support that ADH1C Ile may lower the risk of AD and alcohol abuse as well as alcohol-related cirrhosis in pooled populations, with the strongest and most consistent effects in Asians.

  2. 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.

  3. 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

  4. 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.

  5. 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

  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. Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. Genetic and biochemical studies of the mutant proteins.

    PubMed

    Membrillo-Hernandez, J; Echave, P; Cabiscol, E; Tamarit, J; Ros, J; Lin, E C

    2000-10-27

    The multifunctional AdhE protein of Escherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH(2)-terminal region of the AdhE protein is highly homologous to aldehyde:NAD(+) oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe(2+)-dependent ethanol:NAD(+) oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol. The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA. All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 --> Thr substitution in the acetaldehyde:NAD(+) oxidoreductase domain of AdhE. All second stage mutants grew on ethanol with a doubling time of about 90 min, and all of them produced an AdhE(A267T/E568K). Purified AdhE(A267T) and AdhE(A267T/E568K) showed highly elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhE(A267T/E568K) was more thermal stable than AdhE(A267T). Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhE(A267T). PMID:10922373

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. Associations between ADH gene variants and alcohol phenotypes in Dutch adults.

    PubMed

    van Beek, Jenny H D A; Willemsen, Gonneke; de Moor, Marleen H M; Hottenga, Jouke Jan; Boomsma, Dorret I

    2010-02-01

    Recently, Macgregor et al. (2009) demonstrated significant associations of ADH polymorphisms with reactions to alcohol and alcohol consumption measures in an Australian sample. The aim of the present study was to replicate these findings in a Dutch sample. Survey data on alcohol phenotypes came from 1,754 unrelated individuals registered with the Netherlands Twin Register. SNPs in the ADH gene cluster located on chromosome 4q (n = 491) were subdivided in seven gene sets: ADH5, ADH4, ADH6, ADH1A, ADH1B, ADH1C and ADH7. Within these sets associations of SNPs with alcohol consumption measures, age at onset variables, reactions to alcohol and problem drinking liability were examined. Of the original 38 SNPs studied by Macgregor et al. (2009), six SNPs were not available in our dataset, because one of them had a minor allele frequency < .01 (rs1229984) and five could not be imputed. The remaining SNP associations with alcohol phenotypes as identified by Macgregor et al. (2009) were not replicated in the Dutch sample, after correcting for multiple genotype and phenotype testing. Significant associations were found however, for reactions to alcohol with a SNP in ADH5 (rs6827292, p = .001) and a SNP just upstream of ADH5 (rs6819724, p = .0007) that is in strong LD with rs6827292. Furthermore, an association between age at onset of regular alcohol use and a SNP just upstream of ADH7 (rs2654849, p = .003) was observed. No significant associations were found for alcohol consumption and problem drinking liability. Although these findings do not replicate the earlier findings at the SNP level, the results confirm the role of the ADH gene cluster in alcohol phenotypes.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Combination of ADH1B*2/ALDH2*2 polymorphisms alters acetaldehyde-derived DNA damage in the blood of Japanese alcoholics.

    PubMed

    Yukawa, Yoshiyuki; Muto, Manabu; Hori, Kimiko; Nagayoshi, Haruna; Yokoyama, Akira; Chiba, Tsutomu; Matsuda, Tomonari

    2012-09-01

    The acetaldehyde associated with alcoholic beverages is an evident carcinogen for the esophagus. Genetic polymorphisms of the alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes are associated with the risk of esophageal cancer. However, the exact mechanism via which these genetic polymorphisms affect esophageal carcinogenesis has not been elucidated. ADH1B*2 is involved in overproduction of acetaldehyde due to increased ethanol metabolism into acetaldehyde, and ALDH2*2 is involved in accumulation of acetaldehyde due to the deficiency of acetaldehyde metabolism. Acetaldehyde can interact with DNA and form DNA adducts, resulting in DNA damage. N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) is the most abundant DNA adduct derived from acetaldehyde. Therefore, we quantified N(2)-ethylidene-dG levels in blood samples from 66 Japanese alcoholic patients using liquid chromatography/electrospray tandem mass spectrometry, and investigated the relationship between N(2)-ethylidene-dG levels and ADH1B and ALDH2 genotypes. The median N(2)-ethylidene-dG levels (25th percentile, 75th percentile) in patients with ADH1B*1/*1 plus ALDH2*1/*1, ADH1B*2 carrier plus ALDH2*1/*1, ADH1B*1/*1 plus ALDH2*1/*2, and ADH1B*2 carrier plus ALDH2*1/*2 were 2.14 (0.97, 2.37)/10(7) bases, 2.38 (1.18, 2.98)/10(7) bases, 5.38 (3.19, 6.52)/10(7) bases, and 21.04 (12.75, 34.80)/10(7) bases, respectively. In the ALDH2*1/*2 group, N(2)-ethylidene-dG levels were significantly higher in ADH1B*2 carriers than in the ADH1B*1/*1 group (P < 0.01). N(2)-ethylidene-dG levels were significantly higher in the ALDH2*1/*2 group than in the ALDH2*1/*1 group, regardless of ADH1B genotype (ADH1B*1/*1, P < 0.05; ADH1B*2 carriers, P < 0.01) N(2)-ethylidene-dG levels in blood DNA of the alcoholics was remarkably higher in individuals with a combination of the ADH1B*2 and ALDH2*2 alleles. These results provide a new perspective on the carcinogenicity of the acetaldehyde associated with

  6. [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

  7. 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

  8. 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

  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. 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. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. The Impact of ADH1B Alleles and Educational Status on Levels and Modes of Alcohol Consumption in Russian Male Individuals.

    PubMed

    Borinskaya, S A; Kim, A A; Rubanovich, A V; Yankovsky, N K

    2013-07-01

    Alcohol abuse is one of the main reasons behind the low life span in Russia. Both social and genetic factors affect the alcohol consumption level. The genetic factors are alleles of the alcohol dehydrogenase ADH1B and aldehyde dehydrogenaseALDH2 genes. We have typed and found frequencies for the alleles in a cohort of 642 men, ethnic Russians. The individuals of the cohort were asked to complete a questionnaire in the framework of the Izhevsk Family Study (Leon et al., 2007, 2009) regarding the amount of alcohol consumed and on the type of hazardous alcohol consumption (nonbeverage alcohol consumption and the so-called "zapoï" which is a Russian term for a heavy drinking bout lasting for at least 2 days, when an individual is withdrawn from the normal social life). The ADH1B*48His allele was found among heterozygous individuals only (N=68, 10.6% of the cohort). The ALDH2*504Lys allele was also found among heterozygous individuals only (N=2, 0.3%) The effect of ADH1B alleles and the influence of the education level on the amount and type of alcohol consumed had not previously been studied in Russians. We have found that the amount of consumed alcohol is 21.6% lower (1733 g of ethanol per year) for ADH1B*48His allele carriers in the cohort of Russian men. The amount of consumed alcohol was found to be 9.8% lower (793 g of ethanol per year) in the case when individuals had a higher education as compared to those who had a secondary- or elementary school education level in the same cohort. Hence, the protective effect of the genetic factor (ADH1B*48His allele carriage) has proven to be more pronounced than the influence of the social factor (education level) at the individual level in the cohort of Russian men. Both factors have also proven to have a protective effect against hazardous types of alcohol consumption. Zapoï was not scored among individuals of the cohort with ADH1B*48His allele carriage (OR=12.6, P=0.006), as compared to 8.4% of "zapoï" individuals who

  3. Subjective Response to Alcohol and ADH Polymorphisms in a Select Sample of Young Adult Male East Indians and Africans in Trinidad and Tobago

    PubMed Central

    Montane Jaime, Lazara Karelia; Shafe, Samuel; Liang, Tiebing; Wills, Derek N; Berg, Greta I; Ehlers, Cindy L

    2014-01-01

    Objective: Level of response to alcohol has been associated with risk of alcohol dependence in a number of ethnic groups. In the present study, subjective and objective responses to alcohol were evaluated in Indo-Trinidadians (Indo-T) and Afro-Trinidadians (Afro-T). Associations of alcohol dehydrogenase polymorphisms with response to alcohol, using the Subjective High Assessment Scale (SHAS), and breath alcohol concentrations (BrAC) were tested. Method: Regular male drinkers without alcohol dependence (n = 112) ages 18–25 years participated in alcohol challenge sessions consisting of placebo and two doses of alcohol (target BrAC: 0 g/dl for placebo, .04 g/dl low dose, and .08 g/dl high dose) and genotyped for variants in ADH1B*3 and ADH1C*2. Results: Indo-T had significantly higher BrAC, pulse rates, and cortisol levels when compared with Afro-T but did not have significantly higher SHAS values. Higher responses on the SHAS items muddle/confused and nauseated were significantly associated with the presence of at least one ADH1B*3 allele following the high dose of alcohol in Afro-T. Indo-T with at least one ADH1C*2 allele displayed significantly different Drug × Time interactions for the SHAS item effects of alcohol at the low dose and for the SHAS items clumsy, muddle/confused, effects of alcohol, floating, drunk, and total at the high dose from Indo-T with two ADH1C*1 alleles. Conclusions: This is the first study that has investigated individual sensitivity to alcohol in a Caribbean population and in people of East Indian descent. Indo-T with at least one ADH1C*2 allele may be at higher risk for heavy drinking by feeling less of the effects of alcohol, including nausea. In Afro-T, having at least one ADH1B*3 allele appears to exert a protective effect by enhancing the unpleasant effects of alcohol, such as nausea and confusion. PMID:25208201

  4. 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.

  5. 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

  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. 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.

  8. 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

  9. Meta-Analyses of ALDH2 and ADH1B with Alcohol Dependence in Asians

    ERIC Educational Resources Information Center

    Luczak, Susan E.; Glatt, Stephen J.; Wall, Tamara J.

    2006-01-01

    Meta-analyses were conducted to determine the magnitude of relationships between polymorphisms in 2 genes, ALDH2 and ADH1B, with alcohol dependence in Asians. For each gene, possession of 1 variant [asterisk]2 allele was protective against alcohol dependence, and possession of a 2nd [asterisk]2 allele did not offer significant additional…

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population

    PubMed Central

    Méndez, Claudia

    2015-01-01

    Objective: Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. Methods: ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Results: Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. Conclusions: This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favourable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2 * 2, CYP2E1 * 1 combined with genotype homozygous ALDH2 * 1 found in this study could be leading to the population to a potential risk to alcoholism. PMID:26848198

  20. 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 ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver primary ADH

  1. 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

  2. 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

  3. 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.

  4. Extended genetic effects of ADH cluster genes on the risk of alcohol dependence: from GWAS to replication.

    PubMed

    Park, Byung Lae; Kim, Jee Wook; Cheong, Hyun Sub; Kim, Lyoung Hyo; Lee, Boung Chul; Seo, Cheong Hoon; Kang, Tae-Cheon; Nam, Young-Woo; Kim, Goon-Bo; Shin, Hyoung Doo; Choi, Ihn-Geun

    2013-06-01

    Alcohol dependence (AD) is a multifactorial and polygenic disorder involving complex gene-to-gene and gene-to-environment interactions. Several genome-wide association studies have reported numerous risk factors for AD, but replication results following these studies have been controversial. To identify new candidate genes, the present study used GWAS and replication studies in a Korean cohort with AD. Genome-wide association analysis revealed that two chromosome regions on Chr. 4q22-q23 (ADH gene cluster, including ADH5, ADH4, ADH6, ADH1A, ADH1B, and ADH7) and Chr. 12q24 (ALDH2) showed multiple association signals for the risk of AD. To investigate detailed genetic effects of these ADH genes on AD, a follow-up study of the ADH gene cluster on 4q22-q23 was performed. A total of 90 SNPs, including ADH1B rs1229984 (H47R), were genotyped in an additional 975 Korean subjects. In case-control analysis, ADH1B rs1229984 (H47R) showed the most significant association with the risk of AD (p = 2.63 × 10(-21), OR = 2.35). Moreover, subsequent conditional analyses revealed that all positive associations of other ADH genes in the cluster disappeared, which suggested that ADH1B rs1229984 (H47R) might be the sole functional genetic marker across the ADH gene cluster. Our findings could provide additional information on the ADH gene cluster regarding the risk of AD, as well as a new and important insight into the genetic factors associated with AD.

  5. Ascidian and amphioxus Adh genes correlate functional and molecular features of the ADH family expansion during vertebrate evolution.

    PubMed

    Cañestro, Cristian; Albalat, Ricard; Hjelmqvist, Lars; Godoy, Laura; Jörnvall, Hans; Gonzàlez-Duarte, Roser

    2002-01-01

    The alcohol dehydrogenase (ADH) family has evolved into at least eight ADH classes during vertebrate evolution. We have characterized three prevertebrate forms of the parent enzyme of this family, including one from an urochordate (Ciona intestinalis) and two from cephalochordates (Branchiostoma floridae and Branchiostoma lanceolatum). An evolutionary analysis of the family was performed gathering data from protein and gene structures, exon-intron distribution, and functional features through chordate lines. Our data strongly support that the ADH family expansion occurred 500 million years ago, after the cephalochordate/vertebrate split, probably in the gnathostome subphylum line of the vertebrates. Evolutionary rates differ between the ancestral, ADH3 (glutathione-dependent formaldehyde dehydrogenase), and the emerging forms, including the classical alcohol dehydrogenase, ADH1, which has an evolutionary rate 3.6-fold that of the ADH3 form. Phylogenetic analysis and chromosomal mapping of the vertebrate Adh gene cluster suggest that family expansion took place by tandem duplications, probably concurrent with the extensive isoform burst observed before the fish/tetrapode split, rather than through the large-scale genome duplications also postulated in early vertebrate evolution. The absence of multifunctionality in lower chordate ADHs and the structures compared argue in favor of the acquisition of new functions in vertebrate ADH classes. Finally, comparison between B. floridae and B. lanceolatum Adhs provides the first estimate for a cephalochordate speciation, 190 million years ago, probably concomitant with the beginning of the drifting of major land masses from the Pangea.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. The plant ADH gene family.

    PubMed

    Strommer, Judith

    2011-04-01

    The structures, evolution and functions of alcohol dehydrogenase gene families and their products have been scrutinized for half a century. Our understanding of the enzyme structure and catalytic activity of plant alcohol dehydrogenase (ADH-P) is based on the vast amount of information available for its animal counterpart. The probable origins of the enzyme from a simple β-coil and eventual emergence from a glutathione-dependent formaldehyde dehydrogenase have been well described. There is compelling evidence that the small ADH gene families found in plants today are the survivors of multiple rounds of gene expansion and contraction. To the probable original function of their products in the terminal reaction of anaerobic fermentation have been added roles in yeast-like aerobic fermentation and the production of characteristic scents that act to attract animals that serve as pollinators or agents of seed dispersal and to protect against herbivores.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Genetic Association and Gene-Gene Interaction Reveal Genetic Variations in ADH1B, GSTM1 and MnSOD Independently Confer Risk to Alcoholic Liver Diseases in India

    PubMed Central

    Mukhopadhyay, Indranil; Chatterjee, Ankita; Das, Kausik; Bhowmik, Pradip; Das, Soumyajit; Basu, Priyadarshi; Santra, Amal K.; Datta, Simanti; Dhali, Gopal Krishna; Chowdhury, Abhijit; Banerjee, Soma

    2016-01-01

    Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase (ADH) gene clusters (ADH1B and ADH1C) and Aldehyde dehydrogenase (ALDH2)], one microsomal ethanol oxidizing enzyme cytochrome p450 (CYP2E1) and three oxidative stress response (OSR) genes (MnSOD, GSTT1 and GSTM1) among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes (rs2066701CC in ADH1B, rs1693425TT in ADH1C, rs4880TT in MnSOD and GSTT1/GSTM1 null, p-value <0.05) were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and GSTM1 null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of ADH1C, MnSOD and GSTM1 can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics. PMID:26937962

  18. Genetic Association and Gene-Gene Interaction Reveal Genetic Variations in ADH1B, GSTM1 and MnSOD Independently Confer Risk to Alcoholic Liver Diseases in India.

    PubMed

    Roy, Neelanjana; Dasgupta, Debanjali; Mukhopadhyay, Indranil; Chatterjee, Ankita; Das, Kausik; Bhowmik, Pradip; Das, Soumyajit; Basu, Priyadarshi; Santra, Amal K; Datta, Simanti; Dhali, Gopal Krishna; Chowdhury, Abhijit; Banerjee, Soma

    2016-01-01

    Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase (ADH) gene clusters (ADH1B and ADH1C) and Aldehyde dehydrogenase (ALDH2)], one microsomal ethanol oxidizing enzyme cytochrome p450 (CYP2E1) and three oxidative stress response (OSR) genes (MnSOD, GSTT1 and GSTM1) among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes (rs2066701CC in ADH1B, rs1693425TT in ADH1C, rs4880TT in MnSOD and GSTT1/GSTM1 null, p-value <0.05) were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and GSTM1 null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of ADH1C, MnSOD and GSTM1 can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence.

    PubMed

    Callejas-Negrete, Olga Alicia; Torres-Guzmán, Juan Carlos; Padilla-Guerrero, Israel Enrique; Esquivel-Naranjo, Ulises; Padilla-Ballesteros, Maria Fernanda; García-Tapia, Adriana; Schrank, Augusto; Salazar-Solís, Eduardo; Gutiérrez-Corona, Félix; González-Hernández, Gloria Angélica

    2015-03-01

    Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.

  6. 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.

  7. 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

  8. Associations of ADH and ALDH2 gene variation with self report alcohol reactions, consumption and dependence: an integrated analysis

    PubMed Central

    Macgregor, Stuart; Lind, Penelope A.; Bucholz, Kathleen K.; Hansell, Narelle K.; Madden, Pamela A.F.; Richter, Melinda M.; Montgomery, Grant W.; Martin, Nicholas G.; Heath, Andrew C.; Whitfield, John B.

    2009-01-01

    Alcohol dependence (AD) is a complex disorder with environmental and genetic origins. The role of two genetic variants in ALDH2 and ADH1B in AD risk has been extensively investigated. This study tested for associations between nine polymorphisms in ALDH2 and 41 in the seven ADH genes, and alcohol-related flushing, alcohol use and dependence symptom scores in 4597 Australian twins. The vast majority (4296) had consumed alcohol in the previous year, with 547 meeting DSM-IIIR criteria for AD. There were study-wide significant associations (P < 2.3 × 10−4) between ADH1B-Arg48His (rs1229984) and flushing and consumption, but only nominally significant associations (P < 0.01) with dependence. Individuals carrying the rs1229984 G-allele (48Arg) reported a lower prevalence of flushing after alcohol (P = 8.2 × 10−7), consumed alcohol on more occasions (P = 2.7 × 10−6), had a higher maximum number of alcoholic drinks in a single day (P = 2.7 × 10−6) and a higher overall alcohol consumption (P = 8.9 × 10−8) in the previous year than those with the less common A-allele (48His). After controlling for rs1229984, an independent association was observed between rs1042026 (ADH1B) and alcohol intake (P = 4.7 × 10−5) and suggestive associations (P < 0.001) between alcohol consumption phenotypes and rs1693482 (ADH1C), rs1230165 (ADH5) and rs3762894 (ADH4). ALDH2 variation was not associated with flushing or alcohol consumption, but was weakly associated with AD measures. These results bridge the gap between DNA sequence variation and alcohol-related behavior, confirming that the ADH1B-Arg48His polymorphism affects both alcohol-related flushing in Europeans and alcohol intake. The absence of study-wide significant effects on AD results from the low P-value required when testing multiple single nucleotide polymorphisms and phenotypes. PMID:18996923

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.

    PubMed Central

    Yong, T S; Li, E; Clark, D; Stanley, S L

    1996-01-01

    The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem, we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692838

  15. 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

  16. 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

  17. The Response of Maize Seedlings of Different Ages to Hypoxic and Anoxic Stress (Changes in Induction of Adh1 mRNA, ADH Activity, and Survival of Anoxia).

    PubMed Central

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

    1994-01-01

    Previously we showed that there is only a transient induction of alcohol dehydrogenase 1 (Adh1) transcripts and only a small induction of alcohol dehydrogenase (ADH) enzyme activity in root tips of maize (Zea mays L.) seedlings subjected to strict anaerobiosis without prior acclimation by exposure to low O2 (D.L. Andrews, B.G. Cobb, J.R. Johnson, M.C. Drew [1993] Plant Physiol 101: 403-414). Acclimation of root tips of seedlings by low O2 before anoxia appeared to be necessary for full induction of ADH. Here we have examined the effect of seedling age on changes in the protein content, induction of Adh1 transcripts, and ADH enzyme activity in 5-mm root tips, root axes, and shoots of maize (cv TX5855). Their ability to survive anoxia was also recorded. Some seedlings were sparged with 4% O2 for 6 or 18 h (a hypoxic pretreatment) followed by anoxia (sparged with N2) for up to 48 h. Other seedlings were not acclimated before anoxia. In general, younger seedlings had higher initial (aerobic) levels of total protein, Adh1 transcripts, and ADH activity than did seedlings that were 2 d older. For younger seedlings, anoxia alone induced Adh1 transcripts, which reached a peak within 6 to 12 h, whereas ADH activity increased throughout the 48-h treatment. For older seedlings, anoxia caused only a small, transient induction of Adh1 transcripts or ADH activity. For seedlings of either age, hypoxia induced Adh1 transcripts and ADH activity, both of which were increased further by subsequent anoxia in the younger seedlings but to a lesser extent in the older seedlings. Despite differences in ADH activity, roots of seedlings of either age showed a similar resistance to anoxia. Thus, acclimation of maize seedlings to survive anoxia does not appear to be related to induction of high levels of ADH activity. PMID:12232185

  18. 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

  19. 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

  20. Genome-Wide Significant Association between Alcohol Dependence and a Variant in the ADH Gene Cluster

    PubMed Central

    Frank, Josef; Cichon, Sven; Treutlein, Jens; Ridinger, Monika; Mattheisen, Manuel; Hoffmann, Per; Herms, Stefan; Wodarz, Norbert; Soyka, Michael; Zill, Peter; Maier, Wolfgang; Mössner, Rainald; Gaebel, Wolfgang; Dahmen, Norbert; Scherbaum, Norbert; Schmäl, Christine; Steffens, Michael; Lucae, Susanne; Ising, Marcus; Müller-Myhsok, Bertram; Nöthen, Markus M; Mann, Karl; Kiefer, Falk; Rietschel, Marcella

    2011-01-01

    Alcohol dependence (AD) is an important contributory factor to the global burden of disease. The etiology of AD involves both environmental and genetic factors, and the disorder has a heritability of around 50%. The aim of the present study was to identify susceptibility genes for AD by performing a genome-wide association study (GWAS). The sample comprised 1,333 male in-patients with severe DSM-IV AD and 2,168 controls. These included 487 patients and 1,358 controls from a previous GWAS study by our group. All individuals were of German descent. Single marker tests and a polygenic score based analysis to assess the combined contribution of multiple markers with small effects were performed. The SNP rs1789891, which is located between the ADH1B and ADH1C genes, achieved genome-wide significance (p=1.27E–8; OR=1.46). Other markers from this region were also associated with AD, and conditional analyses indicated that these made a partially independent contribution. The SNP rs1789891 is in complete linkage disequilibrium with the functional Arg272Gln variant (p=1.24E–7, OR=1.31) of the ADH1C gene, which has been reported to modify the rate of ethanol oxidation to acetaldehyde in vitro. A polygenic score based approach produced a significant result (p=9.66E–9). This is the first GWAS of AD to provide genome-wide significant support for the role of the ADH gene cluster and to suggest a polygenic component to the etiology of AD. The latter result suggests that many more AD susceptibility genes still await identification. PMID:22004471

  1. Genome-wide significant association between alcohol dependence and a variant in the ADH gene cluster.

    PubMed

    Frank, Josef; Cichon, Sven; Treutlein, Jens; Ridinger, Monika; Mattheisen, Manuel; Hoffmann, Per; Herms, Stefan; Wodarz, Norbert; Soyka, Michael; Zill, Peter; Maier, Wolfgang; Mössner, Rainald; Gaebel, Wolfgang; Dahmen, Norbert; Scherbaum, Norbert; Schmäl, Christine; Steffens, Michael; Lucae, Susanne; Ising, Marcus; Müller-Myhsok, Bertram; Nöthen, Markus M; Mann, Karl; Kiefer, Falk; Rietschel, Marcella

    2012-01-01

    Alcohol dependence (AD) is an important contributory factor to the global burden of disease. The etiology of AD involves both environmental and genetic factors, and the disorder has a heritability of around 50%. The aim of the present study was to identify susceptibility genes for AD by performing a genome-wide association study (GWAS). The sample comprised 1333 male in-patients with severe AD according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, and 2168 controls. These included 487 patients and 1358 controls from a previous GWAS study by our group. All individuals were of German descent. Single-marker tests and a polygenic score-based analysis to assess the combined contribution of multiple markers with small effects were performed. The single nucleotide polymorphism (SNP) rs1789891, which is located between the ADH1B and ADH1C genes, achieved genome-wide significance [P = 1.27E-8, odds ratio (OR) = 1.46]. Other markers from this region were also associated with AD, and conditional analyses indicated that these made a partially independent contribution. The SNP rs1789891 is in complete linkage disequilibrium with the functional Arg272Gln variant (P = 1.24E-7, OR = 1.31) of the ADH1C gene, which has been reported to modify the rate of ethanol oxidation to acetaldehyde in vitro. A polygenic score-based approach produced a significant result (P = 9.66E-9). This is the first GWAS of AD to provide genome-wide significant support for the role of the ADH gene cluster and to suggest a polygenic component to the etiology of AD. The latter result may indicate that many more AD susceptibility genes still await identification.

  2. 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.

  3. 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.

  4. 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

  5. [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.

  6. Transcriptional control of ADH genes in the xylose-fermenting yeast Pichia stipitis

    SciTech Connect

    Cho, J.Y.; Jeffries, T.W. |

    1999-06-01

    The authors studied the expression of the genes encoding group 1 alcohol dehydrogenases (PsADH1 and PsADH2) in the xylose-fermenting yeast Pichia stipitis CBS 6054. The cells expressed PsADH1 approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of PsADH2 were not detectable under either aeration condition. The authors used a PsADH1::lacZ fusion to monitor PsADH1 expression and found that expression increased as oxygen decreased. The level of PsADH1 transcript was expressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of PsADH1, PsCYC1 expression was regressed. These results indicate that oxygen availability regulates PsADH1 expression and that regulation may be mediated by heme. The regulation of PsADH2 expression was also examined in other genetic backgrounds. Disruption of PsADH1 dramatically increased PsADH2 expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of PsADH2 is subject to feedback regulation under these conditions.

  7. Characterization of the Structure and Evolution of the Adh Region of Drosophila Hydei

    PubMed Central

    Menotti-Raymond, M.; Starmer, W. T.; Sullivan, D. T.

    1991-01-01

    Drosophila of the repleta group have a duplication of the gene which encodes alcohol dehydrogenase (ADH). We report the nucleotide sequence of an 8.4-kb region of genomic DNA of Drosophila hydei which includes the entire Adh region. Analysis of this sequence reveals similarity in organization to the Adh region of Drosophila mojavensis and Drosophila mulleri of the mulleri subgroup, with three genes ordered 5' to 3', Adh-ψ, Adh-2, Adh-1. Deletion of a nucleotide in the second codon of each pseudogene suggests that the first Adh duplication occurred before the divergence of the hydei and mulleri subgroups. However, Adh-1 and Adh-2 of D. hydei are significantly more alike than Adh-1 and Adh-2 of D. mojavensis. Models to account for the difference in similarity between the coding genes were tested by orthologous and paralogous comparisons of the extent of sequence divergence. A model which proposes that independent duplication events generated Adh-1 and Adh-2 in the two lineages is supported by these data. The D. hydei pseudogene is transcribed and the transcript is processed in a complex manner. An intron of greater than 6.2 kb exists between the first ``coding'' exon and an upstream exon which is approximately 250 nucleotides in length. PMID:2004708

  8. Transcriptional Control of ADH Genes in the Xylose-Fermenting Yeast Pichia stipitis

    PubMed Central

    Cho, Jae-Yong; Jeffries, Thomas W.

    1999-01-01

    We studied the expression of the genes encoding group I alcohol dehydrogenases (PsADH1 and PsADH2) in the xylose-fermenting yeast Pichia stipitis CBS 6054. The cells expressed PsADH1 approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of PsADH2 were not detectable under either aeration condition. We used a PsADH1::lacZ fusion to monitor PsADH1 expression and found that expression increased as oxygen decreased. The level of PsADH1 transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of PsADH1, PsCYC1 expression was repressed. These results indicate that oxygen availability regulates PsADH1 expression and that regulation may be mediated by heme. The regulation of PsADH2 expression was also examined in other genetic backgrounds. Disruption of PsADH1 dramatically increased PsADH2 expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of PsADH2 is subject to feedback regulation under these conditions. PMID:10347014

  9. 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

  10. 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

  11. 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.

  12. Patterns of Amino Acid Evolution in the Drosophila ananassae Chimeric Gene, siren, Parallel Those of Other Adh-Derived Chimeras

    PubMed Central

    Shih, Hung-Jui; Jones, Corbin D.

    2008-01-01

    siren1 and siren2 are novel alcohol dehydrogenase (Adh)-derived chimeric genes in the Drosophila bipectinata complex. D. ananassae, however, harbors a single homolog of these genes. Like other Adh-derived chimeric genes, siren evolved adaptively shortly after it was formed. These changes likely shifted the catalytic activity of siren. PMID:18780749

  13. 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

  14. 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

  15. 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.

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

    PubMed

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

    2016-10-25

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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Conserved enhancer and silencer elements responsible for differential Adh transcription in Drosophila cell lines.

    PubMed Central

    Ayer, S; Benyajati, C

    1990-01-01

    The distal promoter of Adh is differentially expressed in Drosophila tissue culture cell lines. After transfection with an exogenous Adh gene, there was a specific increase in distal alcohol dehydrogenase (ADH) transcripts in ADH-expressing (ADH+) cells above the levels observed in transfected ADH-nonexpressing (ADH-) cells. We used deletion mutations and a comparative transient-expression assay to identify the cis-acting elements responsible for enhanced Adh distal transcription in ADH+ cells. DNA sequences controlling high levels of distal transcription were localized to a 15-base-pair (bp) region nearly 500 bp upstream of the distal RNA start site. In addition, a 61-bp negative cis-acting element was found upstream from and adjacent to the enhancer. When this silencer element was deleted, distal transcription increased only in the ADH+ cell line. These distant upstream elements must interact with the promoter elements, the Adf-1-binding site and the TATA box, as they only influenced transcription when at least one of these two positive distal promoter elements was present. Internal deletions targeted to the Adf-1-binding site or the TATA box reduced transcription in both cell types but did not affect the transcription initiation site. Distal transcription in transfected ADH- cells appears to be controlled primarily through these promoter elements and does not involve the upstream regulatory elements. Evolutionary conservation in distantly related Drosophila species suggests the importance of these upstream elements in correct developmental and tissue-specific expression of ADH. Images PMID:1694013

  6. Variation in the ADH1B proximal promoter affects expression.

    PubMed

    Pochareddy, Sirisha; Edenberg, Howard J

    2011-05-30

    The primary pathway of metabolism of dietary alcohol is via its oxidation in liver by alcohol dehydrogenases (ADH). Differences in the ADH enzyme activity or levels of enzyme present could affect the risk for alcoholism. Regulatory variations have been shown to affect the promoter activity and thereby affect the risk for alcoholism. In this study the functional effects of the two SNPs (rs1159918 and rs1229982) in the proximal promoter region of ADH1B that were associated with alcoholism were explored. We examined the effects of five naturally occurring haplotypes on the promoter activity. We observed that a C to A change at rs1229982 increased promoter activity 1.4-fold.

  7. The Adh-related gene of Drosophila melanogaster is expressed as a functional dicistronic messenger RNA: multigenic transcription in higher organisms.

    PubMed Central

    Brogna, S; Ashburner, M

    1997-01-01

    Essentially all eukaryotic cellular mRNAs are monocistronic, and are usually transcribed individually. Two tandemly arranged Drosophila genes, alcohol dehydrogenase (Adh) and Adh-related (Adhr), are transcribed as a dicistronic transcript. From transcripts initiated from the Adh promoter, two classes of mRNA are accumulated, one is monocistronic and encodes Adh alone, the other is dicistronic and includes the open reading frames of both Adh and Adhr. The dicistronic transcript is found in polysomes and the Adhr protein product is detected by antibody staining. We present evidence that the accumulation of the dicistronic mRNA is controlled at the level of the 3' end processing. PMID:9155028

  8. Increased Variation in Adh Enzyme Activity in Drosophila Mutation-Accumulation Experiment Is Not Due to Transposable Elements at the Adh Structural Gene

    PubMed Central

    Aquadro, C. F.; Tachida, H.; Langley, C. H.; Harada, K.; Mukai, T.

    1990-01-01

    We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region. PMID:1963870

  9. 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.

  10. 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

  11. 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.

  12. 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.

  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. 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.

  16. Variations in ADH and ALDH in Southwest California Indians

    PubMed Central

    Ehlers, Cindy L.

    2007-01-01

    Native Americans as a group have the highest rates of alcohol-related deaths of all ethnicities in the United States; however, it remains unclear how and why a greater proportion of individuals in some Native American communities develop alcohol-related problems and alcohol use disorders (AUDs). One potential factor that can influence responses to alcohol are variations in alcohol-metabolizing enzymes. Researchers have analyzed the frequencies of variants in the alcohol-metabolizing enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in some Native American populations. So far the studies have yielded no evidence that an ALDH2 variant, which has shown protective effects in other populations, is found in either American Indians or Alaska Natives. A variant of the ALDH1 enzyme that is encoded by the ALDH1A1*2 allele, however, was found in a small proportion of a group of Southwest California Indians and had a protective effect against alcoholism in that population. Furthermore, a variant of the ADH1B enzyme that is encoded by the ADH1B*3 allele was found in a similar proportion of Southwest California Indians and also was associated with a protective effect. However, these findings do not explain the high prevalence of alcoholism in the tribes investigated. PMID:17718395

  17. Evidence of Positive Selection on a Class I ADH Locus

    PubMed Central

    Han, Yi; Gu, Sheng; Oota, Hiroki; Osier, Michael V.; Pakstis, Andrew J.; Speed, William C.; Kidd, Judith R.; Kidd, Kenneth K.

    2007-01-01

    The alcohol dehydrogenase (ADH) family of enzymes catalyzes the reversible oxidation of alcohol to acetaldehyde. Seven ADH genes exist in a segment of ∼370 kb on 4q21. Products of the three class I ADH genes that share 95% sequence identity are believed to play the major role in the first step of ethanol metabolism. Because the common belief that selection has operated at the ADH1B*47His allele in East Asian populations lacks direct biological or statistical evidence, we used genomic data to test the hypothesis. Data consisted of 54 single-nucleotide polymorphisms (SNPs) across the ADH clusters in a global sampling of 42 populations. Both the Fst statistic and the long-range haplotype (LRH) test provided positive evidence of selection in several East Asian populations. The ADH1B Arg47His functional polymorphism has the highest Fst of the 54 SNPs in the ADH cluster, and it is significantly above the mean Fst of 382 presumably neutral sites tested on the same 42 population samples. The LRH test that uses cores including that site and extending on both sides also gives significant evidence of positive selection in some East Asian populations for a specific haplotype carrying the ADH1B*47His allele. Interestingly, this haplotype is present at a high frequency in only some East Asian populations, whereas the specific allele also exists in other East Asian populations and in the Near East and Europe but does not show evidence of selection with use of the LRH test. Although the ADH1B*47His allele conveys a well-confirmed protection against alcoholism, that modern phenotypic manifestation does not easily translate into a positive selective force, and the nature of that selective force, in the past and/or currently, remains speculative. PMID:17273965

  18. Genetic Variation in the Expression of ADH in DROSOPHILA MELANOGASTER

    PubMed Central

    Maroni, G.; Laurie-Ahlberg, C. C.; Adams, D. A.; Wilton, A. N.

    1982-01-01

    Several chromosomes derived from natural populations have been identified that affect the expression of alcohol dehydrogenase (ADH). Second chromosomes, which also carry the structural gene Adh, show a great deal of polymorphism of genetic elements that determine how much enzyme protein accumulates. The level of enzyme was measured in third instar larvae, 6-to-8-day-old males and in larval fat bodies and alimentary canals. In general, activities in the different organs and stages are highly correlated with one another. One line was found, however, in which the ADH level in the fat body is more than twice the level one would expect on the basis of the activity in alimentary canal. We have also found evidence of third-chromosome elements that affect the level of ADH. PMID:6816669

  19. 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.

  20. Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis.

    PubMed

    Simon, Francis R; Fortune, John; Iwahashi, Mieko; Sutherland, Eileen

    2002-09-01

    Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH and L-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4 in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4 plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4 is the principal determinant of low ADH activity in male liver.

  1. 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.

  2. Effect of ADH II Deficiency on the Intracellular Redox Homeostasis in Zymomonas mobilis

    PubMed Central

    Galinina, Nina; Lasa, Zane; Strazdina, Inese; Rutkis, Reinis; Kalnenieks, Uldis

    2012-01-01

    Mutant strain of the facultatively anaerobic, ethanol-producing bacterium Zymomonas mobilis, deficient in the Fe-containing alcohol dehydrogenase isoenzyme (ADH II), showed impaired homeostasis of the intracellular NAD(P)H during transition from anaerobic to aerobic conditions, and also in steady-state continuous cultures at various oxygen supplies. At the same time, ADH II deficiency in aerobically grown cells was accompanied by a threefold increase of catalase activity and by about 50% increase of hydrogen peroxide excretion. It is concluded that ADH II under aerobic conditions functions to maintain intracellular redox homeostasis and to protect the cells from endogenous hydrogen peroxide. PMID:22629192

  3. Effect of ADH II deficiency on the intracellular redox homeostasis in Zymomonas mobilis.

    PubMed

    Galinina, Nina; Lasa, Zane; Strazdina, Inese; Rutkis, Reinis; Kalnenieks, Uldis

    2012-01-01

    Mutant strain of the facultatively anaerobic, ethanol-producing bacterium Zymomonas mobilis, deficient in the Fe-containing alcohol dehydrogenase isoenzyme (ADH II), showed impaired homeostasis of the intracellular NAD(P)H during transition from anaerobic to aerobic conditions, and also in steady-state continuous cultures at various oxygen supplies. At the same time, ADH II deficiency in aerobically grown cells was accompanied by a threefold increase of catalase activity and by about 50% increase of hydrogen peroxide excretion. It is concluded that ADH II under aerobic conditions functions to maintain intracellular redox homeostasis and to protect the cells from endogenous hydrogen peroxide.

  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. Protein variation in Adh and Adh-related in Drosophila pseudoobscura. Linkage disequilibrium between single nucleotide polymorphisms and protein alleles.

    PubMed Central

    Schaeffer, S W; Walthour, C S; Toleno, D M; Olek, A T; Miller, E L

    2001-01-01

    A 3.5-kb segment of the alcohol dehydrogenase (Adh) region that includes the Adh and Adh-related genes was sequenced in 139 Drosophila pseudoobscura strains collected from 13 populations. The Adh gene encodes four protein alleles and rejects a neutral model of protein evolution with the McDonald-Kreitman test, although the number of segregating synonymous sites is too high to conclude that adaptive selection has operated. The Adh-related gene encodes 18 protein haplotypes and fails to reject an equilibrium neutral model. The populations fail to show significant geographic differentiation of the Adh-related haplotypes. Eight of 404 single nucleotide polymorphisms (SNPs) in the Adh region were in significant linkage disequilibrium with three ADHR protein alleles. Coalescent simulations with and without recombination were used to derive the expected levels of significant linkage disequilibrium between SNPs and 18 protein haplotypes. Maximum levels of linkage disequilibrium are expected for protein alleles at moderate frequencies. In coalescent models without recombination, linkage disequilibrium decays between SNPs and high frequency haplotypes because common alleles mutate to haplotypes that are rare or that reach moderate frequency. The implication of this study is that linkage disequilibrium mapping has the highest probability of success with disease-causing alleles at frequencies of 10%. PMID:11606543

  6. A Global Perspective on Genetic Variation at the ADH Genes Reveals Unusual Patterns of Linkage Disequilibrium and Diversity

    PubMed Central

    Osier, Michael V.; Pakstis, Andrew J.; Soodyall, Himla; Comas, David; Goldman, David; Odunsi, Adekunle; Okonofua, Friday; Parnas, Josef; Schulz, Leslie O.; Bertranpetit, Jaume; Bonne-Tamir, Batsheva; Lu, Ru-Band; Kidd, Judith R.; Kidd, Kenneth K.

    2002-01-01

    Variants of different Class I alcohol dehydrogenase (ADH) genes have been shown to be associated with an effect that is protective against alcoholism. Previous work from our laboratory has shown that the two sites showing the association are in linkage disequilibrium and has identified the ADH1B Arg47His site as causative, with the ADH1C Ile349Val site showing association only because of the disequilibrium. Here, we describe an initial study of the nature of linkage disequilibrium and genetic variation, in population samples from different regions of the world, in a larger segment of the ADH cluster (including the three Class I ADH genes and ADH7). Linkage disequilibrium across ∼40 kb of the Class I ADH cluster is moderate to strong in all population samples that we studied. We observed nominally significant pairwise linkage disequilibrium, in some populations, between the ADH7 site and some Class I ADH sites, at moderate values and at a molecular distance as great as 100 kb. Our data indicate (1) that most ADH-alcoholism association studies have failed to consider many sites in the ADH cluster that may harbor etiologically significant alleles and (2) that the relevance of the various ADH sites will be population dependent. Some individual sites in the Class I ADH cluster show Fst values that are among the highest seen among several dozen unlinked sites that were studied in the same subset of populations. The high Fst values can be attributed to the discrepant frequencies of specific alleles in eastern Asia relative to those in other regions of the world. These alleles are part of a single haplotype that exists at high (>65%) frequency only in the eastern-Asian samples. It seems unlikely that this haplotype, which is rare or unobserved in other populations, reached such high frequency because of random genetic drift alone. PMID:12050823

  7. The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

    PubMed

    Nguyen, Trinh T M; Iwaki, Aya; Izawa, Shingo

    2015-01-01

    Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin. PMID:26696995

  8. The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress

    PubMed Central

    Nguyen, Trinh T. M.; Iwaki, Aya; Izawa, Shingo

    2015-01-01

    Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin. PMID:26696995

  9. The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

    PubMed

    Nguyen, Trinh T M; Iwaki, Aya; Izawa, Shingo

    2015-01-01

    Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin.

  10. 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.

  11. 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.

  12. 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.

  13. Multiple ADH genes modulate risk for drug dependence in both African- and European-Americans

    PubMed Central

    Luo, Xingguang; Kranzler, Henry R.; Zuo, Lingjun; Wang, Shuang; Schork, Nicholas J.; Gelernter, Joel

    2007-01-01

    Drug dependence (DD) is commonly co-morbid with alcohol dependence (AD). Many studies have also shown common genetic risk factors for these disorders. We previously reported associations of AD with seven alcohol dehydrogenase (ADH ) genes. The present study examines the relationship between these genes and DD. We genotyped 16 markers within the ADH gene cluster and 38 unlinked ancestry-informative markers in a case–control sample of 718 individuals. All markers were consistent with Hardy–Weinberg equilibrium in controls, but some markers showed Hardy–Weinberg disequilibrium in cases (minimal P = 0.002). Genotypes of many markers were associated with DD, both before and after controlling for admixture effects (minimal P < 1.0 × 10−6). Diplotype trend regression analysis showed that ADH5 and ADH6 genotypes, and diplotypes at ADH1A, ADH1B, ADH1C and ADH7 (minimal P = 0.002), were associated with DD in European-Americans and/or African-Americans. This first report of an allelic association of these loci with DD provides new insight into the mechanism of genetic risk for DD. These findings, obtained using a series of powerful and reliable analytic methods, may also help to explain the high rate of co-morbidity between AD and DD. PMID:17185388

  14. 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)

  15. 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

  16. Biology, Genetics, and Environment: Underlying Factors Influencing Alcohol Metabolism.

    PubMed

    Wall, Tamara L; Luczak, Susan E; Hiller-Sturmhöfel, Susanne

    2016-01-01

    Gene variants encoding several of the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), are among the largest genetic associations with risk for alcohol dependence. Certain genetic variants (i.e., alleles)--particularly the ADH1B*2, ADH1B*3, ADH1C*1, and ALDH2*2 alleles--have been associated with lower rates of alcohol dependence. These alleles may lead to an accumulation of acetaldehyde during alcohol metabolism, which can result in heightened subjective and objective effects. The prevalence of these alleles differs among ethnic groups; ADH1B*2 is found frequently in northeast Asians and occasionally Caucasians, ADH1B*3 is found predominantly in people of African ancestry, ADH1C*1 varies substantially across populations, and ALDH2*2 is found almost exclusively in northeast Asians. Differences in the prevalence of these alleles may account at least in part for ethnic differences in alcohol consumption and alcohol use disorder (AUD). However, these alleles do not act in isolation to influence the risk of AUD. For example, the gene effects of ALDH2*2 and ADH1B*2 seem to interact. Moreover, other factors have been found to influence the extent to which these alleles affect a person's alcohol involvement, including developmental stage, individual characteristics (e.g., ethnicity, antisocial behavior, and behavioral undercontrol), and environmental factors (e.g., culture, religion, family environment, and childhood adversity).

  17. Biology, Genetics, and Environment: Underlying Factors Influencing Alcohol Metabolism.

    PubMed

    Wall, Tamara L; Luczak, Susan E; Hiller-Sturmhöfel, Susanne

    2016-01-01

    Gene variants encoding several of the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), are among the largest genetic associations with risk for alcohol dependence. Certain genetic variants (i.e., alleles)--particularly the ADH1B*2, ADH1B*3, ADH1C*1, and ALDH2*2 alleles--have been associated with lower rates of alcohol dependence. These alleles may lead to an accumulation of acetaldehyde during alcohol metabolism, which can result in heightened subjective and objective effects. The prevalence of these alleles differs among ethnic groups; ADH1B*2 is found frequently in northeast Asians and occasionally Caucasians, ADH1B*3 is found predominantly in people of African ancestry, ADH1C*1 varies substantially across populations, and ALDH2*2 is found almost exclusively in northeast Asians. Differences in the prevalence of these alleles may account at least in part for ethnic differences in alcohol consumption and alcohol use disorder (AUD). However, these alleles do not act in isolation to influence the risk of AUD. For example, the gene effects of ALDH2*2 and ADH1B*2 seem to interact. Moreover, other factors have been found to influence the extent to which these alleles affect a person's alcohol involvement, including developmental stage, individual characteristics (e.g., ethnicity, antisocial behavior, and behavioral undercontrol), and environmental factors (e.g., culture, religion, family environment, and childhood adversity). PMID:27163368

  18. 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.

  19. 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.

  20. Identification of Elymus (Triticeae, Poaceae) and its related genera genomes by RFLP analysis of PCR-amplified Adh genes.

    PubMed

    Liu, QuanLan; Zhang, NingNing; Li, Lei; Liu, Jie

    2010-10-01

    Elymus L. is the largest genus in Triticeae, containing about 150 species with four recognized genome donors (St, H, P, and W). Traditionally, the genome compound of this genus is identified based on cytological data. Recently, molecular phylogenetic analysis was used to investigate its genomic combination. Here we describe a restriction fragment length polymorphism (RFLP) assay based on digesting alcohol dehydrogenase (Adh) amplicons with two restriction enzyme combinations, EcoRI-HindIII and EcoRI-PstI, which easily can be used to distinguish Elymus and its closely related genera genomes. The method includes only four steps: (1) amplifying nuclear Adh genes with universal primers; (2) purifying and cloning PCR products; (3) digesting plasmids with restriction enzymes that identify a given genome; (4) running the digested products on an agarose gel and identify the sample based on the restriction profiles. Results showed that: (1) PCR products ranged from 1,200 to 2,000 bp; (2) Adh2 gene was amplified from all the tested genomes; Adh1 gene was amplified from almost all of the tested genomes except the W genome; Adh3 gene was amplified only from the St genome; (3) the EcoRI-HindIII combination was effective to distinguish different Adh gene types (Adh1, Adh2, and Adh3); (4) the Adh2-EcoRI-PstI fragments could be used to distinguish Elymus and its closely related genera genomes. Therefore, This RFLP assay provides an inexpensive and simple means of identifying Elymus genomes.

  1. Ethanol formation in adh0 mutants reveals the existence of a novel acetaldehyde-reducing activity in Saccharomyces cerevisiae.

    PubMed Central

    Drewke, C; Thielen, J; Ciriacy, M

    1990-01-01

    A strain of Saccharomyces cerevisiae has been constructed which is deficient in the four alcohol dehydrogenase (ADH) isozymes known at present. This strain (adh0), being irreversibly mutated in the genes ADH1, ADH3, and ADH4 and carrying a point mutation in the gene ADH2 coding for the glucose-repressible isozyme ADHII, still produces up to one third of the theoretical maximum yield of ethanol in a homofermentative conversion of glucose to ethanol. Analysis of the glucose metabolism of adh0 cells shows that the lack of all known ADH isozymes results in the formation of glycerol as a major fermentation product, accompanied by a significant production of acetaldehyde and acetate. Treatment of glucose-growing adh0 cells with the respiratory-chain inhibitor antimycin A leads to an immediate cessation of ethanol production, demonstrating that ethanol production in adh0 cells is dependent on mitochondrial electron transport. Reduction of acetaldehyde to ethanol in isolated mitochondria could also be demonstrated. This reduction is apparently linked to the oxidation of acetaldehyde to acetate. Preliminary data suggest that this novel type of ethanol formation in S. cerevisiae is associated with the inner mitochondrial membrane. Images PMID:2193925

  2. A highly conserved sequence in the 3'-untranslated region of the drosophila Adh gene plays a functional role in Adh expression.

    PubMed Central

    Parsch, J; Stephan, W; Tanda, S

    1999-01-01

    Phylogenetic analysis identified a highly conserved eight-base sequence (AAGGCTGA) within the 3'-untranslated region (UTR) of the Drosophila alcohol dehydrogenase gene, Adh. To examine the functional significance of this conserved motif, we performed in vitro deletion mutagenesis on the D. melanogaster Adh gene followed by P-element-mediated germline transformation. Deletion of all or part of the eight-base sequence leads to a twofold increase in in vivo ADH enzymatic activity. The increase in activity is temporally and spatially general and is the result of an underlying increase in Adh transcript. These results indicate that the conserved 3'-UTR motif plays a functional role in the negative regulation of Adh gene expression. The evolutionary significance of our results may be understood in the context of the amino acid change that produces the ADH-F allele and also leads to a twofold increase in ADH activity. While there is compelling evidence that the amino acid replacement has been a target of positive selection, the conservation of the 3'-UTR sequence suggests that it is under strong purifying selection. The selective difference between these two sequence changes, which have similar effects on ADH activity, may be explained by different metabolic costs associated with the increase in activity. PMID:9927459

  3. 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

  4. ADH IB expression, but not ADH III, is decreased in human lung cancer.

    PubMed

    Mutka, Sarah C; Green, Lucia H; Verderber, Evie L; Richards, Jane P; Looker, Doug L; Chlipala, Elizabeth A; Rosenthal, Gary J

    2012-01-01

    Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.

  5. ADH IB Expression, but Not ADH III, Is Decreased in Human Lung Cancer

    PubMed Central

    Mutka, Sarah C.; Green, Lucia H.; Verderber, Evie L.; Richards, Jane P.; Looker, Doug L.; Chlipala, Elizabeth A.; Rosenthal, Gary J.

    2012-01-01

    Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer. PMID:23285246

  6. ADH IB expression, but not ADH III, is decreased in human lung cancer.

    PubMed

    Mutka, Sarah C; Green, Lucia H; Verderber, Evie L; Richards, Jane P; Looker, Doug L; Chlipala, Elizabeth A; Rosenthal, Gary J

    2012-01-01

    Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer. PMID:23285246

  7. Ethanol-Induced ADH Activity in Zebrafish: Differential Concentration-Dependent Effects on High- Versus Low-Affinity ADH Enzymes.

    PubMed

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

    2016-04-01

    Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, including humans. We previously demonstrated that acute ethanol exposure increases alcohol dehydrogenase (ADH) activity in an inverted U-shaped dose-dependent manner. It was hypothesized that the biphasic dose-response was due to the increased activity of a high-affinity ADH isoform following exposure to low concentrations of ethanol and increased activity of a low-affinity ADH isoform following exposure to higher concentrations of ethanol. To test this hypothesis, we exposed zebrafish to different concentrations of ethanol (0%, 0.25%, 0.5%, and 1.0% v/v) for 30 min and measured the total ADH activity in the zebrafish liver. However, we also repeated this enzyme activity assay using a low concentration of the substrate (ethanol) to determine the activity of high-affinity ADH isoforms. We found that total ADH activity in response to ethanol induces an inverted U-shaped dose-response similar to our previous study. Using a lower substrate level in our enzyme assay targeting high-affinity isozymes, we found a similar dose-response. However, the difference in activity between the high and low substrate assays (high substrate activity - low substrate activity), which provide an index of activity for low-affinity ADH isoforms, revealed no significant effect of ethanol exposure. Our results suggest that the inverted U-shaped dose-response for total ADH activity in response to ethanol is driven primarily by high-affinity isoforms of ADH.

  8. The Joint Effects of ADH1B Variants and Childhood Adversity on Alcohol-Related Phenotypes in African-American and European-American Women and Men

    PubMed Central

    Sartor, Carolyn E.; Wang, Zuoheng; Xu, Ke; Kranzler, Henry R.; Gelernter, Joel

    2015-01-01

    Background The ADH1B gene has consistently been implicated in problem drinking, but rarely incorporated into gene by environment investigations of alcohol phenotypes. This study examined the joint effects of variation in ADH1B and childhood adversity – a well-documented risk factor for alcohol problems and moderator of genetic liability to psychiatric outcomes – on maximum drinks consumed in a 24-hour period (maxdrinks) and alcohol use disorder (AUD) symptoms. Methods Data were drawn from 2,617 African-American (AA) and 1,436 European-American (EA) participants (42% female) in a multisite genetic study of substance dependence. We tested the most significant ADH1B SNPs for alcohol dependence from a genomewide association study with this sample, ADH1B-rs1229984 (Arg48His) and ADH1B-rs2066702 (Arg370Cys), in EA and AA subsamples, respectively. Results Ordinal regression analyses conducted separately by sex and population revealed significant main effects for childhood adversity both for alcohol phenotypes in AA women and men and for maxdrinks in EA women. A significant rs1229984 by childhood adversity interaction was observed for AUD symptoms in EA men. Unexposed His-allele carriers reported a mean of 3.6 AUD criteria, but adversity-exposed His-allele carriers endorsed approximately the same number (6.3) as those without the protective allele (6.3 and 7.0 for adversity-exposed and adversity-unexposed groups, respectively). Conclusions Results suggest that under conditions of childhood adversity, the His allele does not exert its protective effects in EA men (OR=0.57, CI:0.32–1.01; p=0.056). Findings highlight the robust risk effect conferred by childhood adversity and the importance of considering population and sex in genetically informative investigations of its association with alcohol outcomes. PMID:25410943

  9. 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.

  10. 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.

  11. Improving the NADH-cofactor specificity of the highly active AdhZ3 and AdhZ2 from Escherichia coli K-12.

    PubMed

    Pick, André; Ott, Wolfgang; Howe, Thomas; Schmid, Jochen; Sieber, Volker

    2014-11-10

    Biocatalysis is a promising tool for the sustainable production of chemicals. When cofactor depending enzymatic reactions are involved the applicability of the right cofactor is a central issue. One important example in this regard is the production of alcohols by nicotinamide cofactor (NAD(P)(+)) depending alcohol dehydrogenases. AdhZ3 from Escherichia coli, which is important for the production of alcohols from biomass, has a preference for NADPH as cofactor. We used a structure guided site-specific random approach, to change the cofactor preference towards NADH and to deduce more general rules for redesigning the cofactor specificity. Transfer of a triplet motif from NADH preferring horse liver ADH to AdhZ3 showed an insufficient switch in the preference towards NADH. A combinatorial site saturation mutagenesis altering three residues at once was applied. Library screening with two different cofactor concentrations (0.1 and 0.3mM) resulted in nine improved variants with AdhZ3-LND having the highest vmax and AdhZ3-CND having the lowest K(m). Asparagine was the most frequent amino acid found in eight of nine triplet motifs. To verify the triplet-motif, two variants of E. coli AdhZ2 DIN and LND were designed and confirmed for improved activity with NADH.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Fusarium oxysporum Adh1 has dual fermentative and oxidative functions and is involved in fungal virulence in tomato plants.

    PubMed

    Corrales Escobosa, Alma Rosa; Rangel Porras, Rosa Angelica; Meza Carmen, Victor; Gonzalez Hernandez, Gloria Angélica; Torres Guzman, Juan Carlos; Wrobel, Kazimierz; Wrobel, Katarzyna; Roncero, M Isabel G; Gutierrez Corona, J Felix

    2011-09-01

    An alcohol dehydrogenase gene, adh1, has been identified in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that adh1 is highly expressed in mycelia grown in potato dextrose liquid medium (PDB) under hypoxic conditions, as compared to mycelia grown under aerobic conditions. One spontaneous allyl alcohol-resistant (Ally(R)) mutant exhibited insertion of an incomplete F.oxysporum transposable element, while another mutant contained a short (13 nucleotide) deletion, in both cases interrupting the coding region of the adh1 gene. These mutations caused deficiency in Adh activity due to loss of the main constitutive isoform of Adh1, as well as alteration of different physiological parameters related to carbon and energy metabolism, including the ability to use ethanol as a carbon source under aerobic conditions; impaired growth under hypoxic conditions with glucose as the carbon source; and diminished production of ethanol in glucose-containing medium. Interestingly, the adh1 mutations resulted in a significant delay in fungal disease development in tomato plants. Complementation with the wild-type adh1 allele repaired all defects caused by mutation, indicating that the product of the adh1 gene has dual enzymatic functions (fermentative and oxidative), depending on culture conditions, and is also required for full fungal virulence.

  2. [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.

  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. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Alcohol-induced insulin resistance in liver: Potential roles in regulation of ADH expression; ethanol clearance and alcohol liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using total enteral nutrition (TEN), we demonstrated that low carbohydrate, high alcohol-containing diets (10-12 g/kg/dO produced alcoholic liver disease (ALD) in adult male Sprague-Dawley rats (300 g). Intragastric infusion of this diet generates regular pulses of blood ethanol concentrations (BEC...

  13. The ADH gene cluster SNP rs1789891 and temperamental dimensions in patients with alcohol dependence and affective disorders.

    PubMed

    Oniszczenko, Włodzimierz; Rybakowski, Janusz K; Dragan, Wojciech Ł; Grzywacz, Anna; Samochowiec, Jerzy

    2015-08-01

    This study had three objectives: (1) to assess the relationship between the single nucleotide polymorphism (SNP) rs1789891 in the alcohol dehydrogenase gene cluster and alcohol dependence and affective disorders; (2) to assess the differences in the Regulative Theory of Temperament (RTT) traits between an alcohol dependent group, an affective disorders group, and a healthy group; and (3) to assess the relationship between rs1789891 and temperament traits in a healthy group, taking into account the interaction of genotype and sex. The SNP rs1789891 was genotyped in a group of 194 alcohol dependent men, aged 21 to 71 years; 137 patients with affective disorders, including 51 males and 86 females, aged 19 to 85 years; and a group of 207 healthy individuals, including 89 males and 118 females, aged 18 to 71 years. Temperament traits (briskness, perseveration, sensory sensitivity, emotional reactivity, endurance, and activity) were assessed in all groups using the Formal Characteristics of Behaviour-Temperament Inventory. The comparative analysis of genotypic frequencies showed no significant differences between patients with alcoholism or affective disorders and those in the control group. Alcohol dependent men and the affective disorder group were characterised by higher levels of emotional reactivity (p-value 1.4e-5 and 9.84e-7, respectively) and lower levels of briskness, sensory sensitivity, endurance, and activity (p-value from 3.76e-8 to 0.012) when compared to the healthy group. The rs1789891 polymorphism was associated with briskness (p = 0.02), sensory sensitivity (p = 0.036), and activity (p = 0.049). None of the results were statistically significant after Bonferroni correction.

  14. Ethnic Related Selection for an ADH Class I Variant within East Asia

    PubMed Central

    Li, Hui; Gu, Sheng; Cai, Xiaoyun; Speed, William C.; Pakstis, Andrew J.; Golub, Efim I.; Kidd, Judith R.; Kidd, Kenneth K.

    2008-01-01

    Background The alcohol dehydrogenases (ADH) are widely studied enzymes and the evolution of the mammalian gene cluster encoding these enzymes is also well studied. Previous studies have shown that the ADH1B*47His allele at one of the seven genes in humans is associated with a decrease in the risk of alcoholism and the core molecular region with this allele has been selected for in some East Asian populations. As the frequency of ADH1B*47His is highest in East Asia, and very low in most of the rest of the world, we have undertaken more detailed investigation in this geographic region. Methodology/Principal Findings Here we report new data on 30 SNPs in the ADH7 and Class I ADH region in samples of 24 populations from China and Laos. These populations cover a wide geographic region and diverse ethnicities. Combined with our previously published East Asian data for these SNPs in 8 populations, we have typed populations from all of the 6 major linguistic phyla (Altaic including Korean-Japanese and inland Altaic, Sino-Tibetan, Hmong-Mien, Austro-Asiatic, Daic, and Austronesian). The ADH1B genotyping data are strongly related to ethnicity. Only some eastern ethnic phyla or subphyla (Korean-Japanese, Han Chinese, Hmong-Mien, Daic, and Austronesian) have a high frequency of ADH1B*47His. ADH1B haplotype data clustered the populations into linguistic subphyla, and divided the subphyla into eastern and western parts. In the Hmong-Mien and Altaic populations, the extended haplotype homozygosity (EHH) and relative EHH (REHH) tests for the ADH1B core were consistent with selection for the haplotype with derived SNP alleles. In the other ethnic phyla, the core showed only a weak signal of selection at best. Conclusions/Significance The selection distribution is more significantly correlated with the frequency of the derived ADH1B regulatory region polymorphism than the derived amino-acid altering allele ADH1B*47His. Thus, the real focus of selection may be the regulatory region

  15. 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.

  16. [Verification and Validation on Single Nucleotide Polymorphism Analysis of Alcohol Metabolism-Related Genes ADH1B and ALDH2, Using Dried-Saliva Samples].

    PubMed

    Murata, Shigenori; Hayashida, Mariko; Ishiguro-Tanaka, Yuko; Imazeki, Hiromi; Hayashi, Emiko; Yokoyama, Akira; Kinoshita, Kenji

    2015-11-01

    We have developed a new method for unprocessed biological specimens as templates directly into the TaqMan assay. Saliva was needed to be put on a water-soluble paper and dried, because foreign substances, such as a filter paper, hinder fluorescence detection through the assay. Genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) polymorphisms was, subsequently, performed by TaqMan PCR assay using dried saliva in the present investigation. The optimized technique was tested on 114 samples of alcoholic patients. The PCR-RFLP methods with purified DNA from blood samples were employed for validation of the assay. Upon validation, complete concordance was observed between the two independent results. These results highlight the ability of TaqMan PCR assays using dried saliva on water-soluble paper in genotyping of ADH1B and ALDH2 genes. Our results showed a rapid, simple, reliable, and cost-effective method for SNP genotyping of mutations in ADH1B and ALDH2 genes. This will be very useful for large-scale association studies in various fields. [Original]. PMID:26995869

  17. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati.

    PubMed

    Yaacob, Norhayati; Mohamad Ali, Mohd Shukuri; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  18. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati

    PubMed Central

    Yaacob, Norhayati; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  19. Medium-chain and short-chain dehydrogenases/reductases in retinoid metabolism

    PubMed Central

    Parés, X.; Farrés, J.; Kedishvili, N.; Duester, G.

    2009-01-01

    Retinoic acid (RA), the most active retinoid, is synthesized in two steps from retinol. The first step, oxidation of retinol to retinaldehyde, is catalyzed by cytosolic alcohol dehydrogenases (ADHs) of the medium-chain dehydrogenase/reductase (MDR) superfamily and microsomal retinol dehydrogenases (RDHs) of the short-chain dehydrogenase/reductase (SDR) superfamily. The second step, oxidation of retinaldehyde to RA, is catalyzed by several aldehyde dehydrogenases. ADH1 and ADH2 are the major MDR enzymes in liver retinol detoxification, while ADH3 (less active) and ADH4 (most active) participate in RA generation in tissues. Several NAD+- and NADP+-dependent SDRs are retinoid active. Their in vivo contribution has been demonstrated in the visual cycle (RDH5, RDH12), adult retinoid homeostasis (RDH1) and embryogenesis (RDH10). Km values for most retinoid-active ADHs and RDHs are close to 1 μM or lower, suggesting that they participate physiologically in retinol/retinaldehyde interconversion. Probably none of these enzymes uses retinoids bound to cellular retinol-binding protein, but only free retinoids. The large number of enzymes involved in the two directions of this step, also including aldoketo reductases, suggests that retinaldehyde levels are strictly regulated. PMID:19011747

  20. 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

  1. 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.

  2. The Relationship between CmADHs and the Diversity of Volatile Organic Compounds of Three Aroma Types of Melon (Cucumis melo)

    PubMed Central

    Chen, Hao; Cao, Songxiao; Jin, Yazhong; Tang, Yufan; Qi, Hongyan

    2016-01-01

    Alcohol dehydrogenase (ADH) plays an important role in aroma volatile compounds synthesis of plants. In this paper, we tried to explore the relationship between CmADHs and the volatile organic compounds (VOCs) in oriental melon. Three different aroma types of melon were used as materials. The principle component analysis of three types of melon fruit was conducted. We also measured the CmADHs expression level and enzymatic activities of ADH and alcohol acyl-transferase (AAT) on different stages of fruit ripening. An incubation experiment was carried out to investigate the effect of substrates and inhibitor (4-MP, 4-methylpyrazole) on CmADHs expression, ADH activity, and the main compounds of oriental melon. The results illustrated that ethyl acetate, hexyl acetate (E,Z)-3,6-nonadien-1-ol and 2-ethyl-2hexen-1-ol were the four principal volatile compounds of these three types of melon. AAT activity was increasing with fruit ripening, and the AAT activity in CH were the highest, whereas ADH activity peaked on 32 DAP, 2 days before maturation, and the ADH activity in CB and CG were higher than that in CH. The expression pattern of 11 CmADH genes from 24 to 36 day after pollination (DAP) was found to vary in three melon varieties. CmADH4 was only expressed in CG and the expression levels of CmADH3 and CmADH12 in CH and CB were much higher than that in CG, and they both peaked 2 days before fruit ripening. Ethanol and 4-MP decreased the reductase activity of ADH, the expression of most CmADHs and ethyl acetate or hexyl acetate contents of CB, except for 0.1 mM 4-MP, while aldehyde improved the two acetate ester contents. In addition, we found a positive correlation between the expression of CmADH3 and CmADH12 and the key volatile compound of CB. The relationship between CmADHs and VOCs synthesis of oriental melon was discussed. PMID:27445845

  3. The Relationship between CmADHs and the Diversity of Volatile Organic Compounds of Three Aroma Types of Melon (Cucumis melo).

    PubMed

    Chen, Hao; Cao, Songxiao; Jin, Yazhong; Tang, Yufan; Qi, Hongyan

    2016-01-01

    Alcohol dehydrogenase (ADH) plays an important role in aroma volatile compounds synthesis of plants. In this paper, we tried to explore the relationship between CmADHs and the volatile organic compounds (VOCs) in oriental melon. Three different aroma types of melon were used as materials. The principle component analysis of three types of melon fruit was conducted. We also measured the CmADHs expression level and enzymatic activities of ADH and alcohol acyl-transferase (AAT) on different stages of fruit ripening. An incubation experiment was carried out to investigate the effect of substrates and inhibitor (4-MP, 4-methylpyrazole) on CmADHs expression, ADH activity, and the main compounds of oriental melon. The results illustrated that ethyl acetate, hexyl acetate (E,Z)-3,6-nonadien-1-ol and 2-ethyl-2hexen-1-ol were the four principal volatile compounds of these three types of melon. AAT activity was increasing with fruit ripening, and the AAT activity in CH were the highest, whereas ADH activity peaked on 32 DAP, 2 days before maturation, and the ADH activity in CB and CG were higher than that in CH. The expression pattern of 11 CmADH genes from 24 to 36 day after pollination (DAP) was found to vary in three melon varieties. CmADH4 was only expressed in CG and the expression levels of CmADH3 and CmADH12 in CH and CB were much higher than that in CG, and they both peaked 2 days before fruit ripening. Ethanol and 4-MP decreased the reductase activity of ADH, the expression of most CmADHs and ethyl acetate or hexyl acetate contents of CB, except for 0.1 mM 4-MP, while aldehyde improved the two acetate ester contents. In addition, we found a positive correlation between the expression of CmADH3 and CmADH12 and the key volatile compound of CB. The relationship between CmADHs and VOCs synthesis of oriental melon was discussed. PMID:27445845

  4. The Relationship between CmADHs and the Diversity of Volatile Organic Compounds of Three Aroma Types of Melon (Cucumis melo).

    PubMed

    Chen, Hao; Cao, Songxiao; Jin, Yazhong; Tang, Yufan; Qi, Hongyan

    2016-01-01

    Alcohol dehydrogenase (ADH) plays an important role in aroma volatile compounds synthesis of plants. In this paper, we tried to explore the relationship between CmADHs and the volatile organic compounds (VOCs) in oriental melon. Three different aroma types of melon were used as materials. The principle component analysis of three types of melon fruit was conducted. We also measured the CmADHs expression level and enzymatic activities of ADH and alcohol acyl-transferase (AAT) on different stages of fruit ripening. An incubation experiment was carried out to investigate the effect of substrates and inhibitor (4-MP, 4-methylpyrazole) on CmADHs expression, ADH activity, and the main compounds of oriental melon. The results illustrated that ethyl acetate, hexyl acetate (E,Z)-3,6-nonadien-1-ol and 2-ethyl-2hexen-1-ol were the four principal volatile compounds of these three types of melon. AAT activity was increasing with fruit ripening, and the AAT activity in CH were the highest, whereas ADH activity peaked on 32 DAP, 2 days before maturation, and the ADH activity in CB and CG were higher than that in CH. The expression pattern of 11 CmADH genes from 24 to 36 day after pollination (DAP) was found to vary in three melon varieties. CmADH4 was only expressed in CG and the expression levels of CmADH3 and CmADH12 in CH and CB were much higher than that in CG, and they both peaked 2 days before fruit ripening. Ethanol and 4-MP decreased the reductase activity of ADH, the expression of most CmADHs and ethyl acetate or hexyl acetate contents of CB, except for 0.1 mM 4-MP, while aldehyde improved the two acetate ester contents. In addition, we found a positive correlation between the expression of CmADH3 and CmADH12 and the key volatile compound of CB. The relationship between CmADHs and VOCs synthesis of oriental melon was discussed.

  5. 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.

  6. 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.

  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. 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

  10. Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

    PubMed Central

    Wu, C. Y.; Mote-Jr., J.; Brennan, M. D.

    1990-01-01

    Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed. PMID:2165967

  11. 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

  12. Contribution of NADH increases to ethanol’s inhibition of retinol oxidation by human ADH isoforms

    PubMed Central

    Chase, Jennifer R.; Poolman, Mark G.; Fell, David A.

    2010-01-01

    Background A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free NADH in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms. Method To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of four reactions: (1) ADH oxidation of ethanol and NAD+ (2) ADH oxidation of retinol and NAD+ (3) oxidation of ethanol by a generalized Ethanoloxidase that uses NAD+ (4) NADHoxidase which carries out NADH turnover. Results Using the metabolic modeling package SCRUMPY, we have shown that the ethanol-induced increase in NADH contributes from 0–90% of the inhibition by ethanol, depending on [ethanol] and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanoloxidase and the NADHoxidase contribute as well. Discussion Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases. PMID:19183134

  13. 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

  14. 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

  15. In vivo introduction of unpreferred synonymous codons into the Drosophila Adh gene results in reduced levels of ADH protein.

    PubMed Central

    Carlini, David B; Stephan, Wolfgang

    2003-01-01

    The evolution of codon bias, the unequal usage of synonymous codons, is thought to be due to natural selection for the use of preferred codons that match the most abundant species of isoaccepting tRNA, resulting in increased translational efficiency and accuracy. We examined this hypothesis by introducing 1, 6, and 10 unpreferred codons into the Drosophila alcohol dehydrogenase gene (Adh). We observed a significant decrease in ADH protein production with number of unpreferred codons, confirming the importance of natural selection as a mechanism leading to codon bias. We then used this empirical relationship to estimate the selection coefficient (s) against unpreferred synonymous mutations and found the value (s >or= 10(-5)) to be approximately one order of magnitude greater than previous estimates from population genetics theory. The observed differences in protein production appear to be too large to be consistent with current estimates of the strength of selection on synonymous sites in D. melanogaster. PMID:12586711

  16. 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

  17. Regulation of Drosophila Adh promoter switching by an initiator-targeted repression mechanism.

    PubMed Central

    Ren, B; Maniatis, T

    1998-01-01

    The stage-specific expression of the Drosophila alcohol dehydrogenase (Adh) gene is achieved through the alternate activation of two tandem promoters. The proximal promoter is active primarily during late embryonic development and early larval stages, while the distal promoter is active in late third instar larvae and adults. Here, we provide evidence that this Adh promoter switch is regulated by a zinc finger repressor protein (AEF-1) that is expressed predominantly in adult flies and targets the initiator region of the proximal promoter. We propose that AEF-1 plays a critical role in Adh promoter switching by blocking interactions between a component of the general transcription machinery and the initiator region of the proximal promoter. PMID:9463385

  18. 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

  19. 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...

  20. 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…

  1. 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.

  2. 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…

  3. 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

  4. Associations between DNA Sequence Variation and Variation in Expression of the Adh Gene in Natural Populations of Drosophila Melanogaster

    PubMed Central

    Laurie, C. C.; Bridgham, J. T.; Choudhary, M.

    1991-01-01

    A large part of the genetic variation in alcohol dehydrogenase (ADH) activity level in natural populations of Drosophila melanogaster is associated with segregation of an amino acid replacement polymorphism at nucleotide 1490, which generates a difference in electrophoretic mobility. Part of the allozymic difference in activity level is due to a catalytic efficiency difference, which is also caused by the amino acid replacement, and part is due to a difference in the concentration of ADH protein. A previous site-directed in vitro mutagenesis experiment clearly demonstrated that the amino acid replacement has no effect on the concentration of ADH protein, nor does a strongly associated silent polymorphism at nucleotide 1443. Here we analyze associations between polymorphisms within the Adh gene and variation in ADH protein level for a number of chromosomes derived from natural populations. A sequence length polymorphism within the first intron of the distal (adult) transcript, &1, is in strong linkage disequilibrium with the amino acid replacement. Among a sample of 46 isochromosomal lines analyzed, all but one of the 14 Fast lines have &1 and all but one of the 32 Slow lines lack &1. The exceptional Fast line has an unusually low level of ADH protein (typical of Slow lines) and the exceptional Slow line has an unusually high level (typical of Fast lines). These results suggest that the &1 polymorphism may be responsible for the average difference in ADH protein between the allozymic classes. A previous experiment localized the effect on ADH protein to a 2.3-kb restriction fragment. DNA sequences of this fragment from several alleles of each allozymic type indicate that no other polymorphisms within this region are as closely associated with the ADH protein level difference as the &1 polymorphism. PMID:1683848

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. Alcohol metabolism by oral streptococci and interaction with human papillomavirus leads to malignant transformation of oral keratinocytes.

    PubMed

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

    2015-01-01

    Poor oral hygiene, ethanol consumption, and human papillomavirus (HPV) are associated with oral and esophageal cancers. However, the mechanism is not fully known. This study examines alcohol metabolism in Streptococcus and its interaction with HPV-16 in the malignant transformation of oral keratinocytes. The acetaldehyde-producing strain Streptococcus gordonii V2016 was analyzed for adh genes and activities of alcohol and aldehyde dehydrogenases. Streptococcus attachment to immortalized HPV-16 infected human oral keratinocytes, HOK (HPV/HOK-16B), human oral buccal keratinocytes, and foreskin keratinocytes was studied. Acetaldehyde, malondialdehyde, DNA damage, and abnormal proliferation among keratinocytes were also quantified. We found 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. S. gordonii V2016 did not show a detectable aldehyde dehydrogenase. AdhE is the major alcohol dehydrogenase in S. gordonii. Acetaldehyde and malondialdehyde production from permissible Streptococcus species significantly increased the bacterial attachment to keratinocytes, which was associated with an enhanced expression of furin to facilitate HPV infection and several malignant phenotypes including acetaldehyde adduct formation, abnormal proliferation, and enhanced migration through integrin-coated basement membrane by HPV-infected oral keratinocytes. Therefore, expression of multiple alcohol dehydrogenases with no functional aldehyde dehydrogenase contributes to excessive production of acetaldehyde from ethanol by oral streptococci. Oral Streptococcus species and HPV may cooperate to transform oral keratinocytes after ethanol exposure. These results suggest a significant clinical interaction, but further validation is warranted. PMID:25427911

  10. Alcohol metabolism by oral streptococci and interaction with human papillomavirus leads to malignant transformation of oral keratinocytes.

    PubMed

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

    2015-01-01

    Poor oral hygiene, ethanol consumption, and human papillomavirus (HPV) are associated with oral and esophageal cancers. However, the mechanism is not fully known. This study examines alcohol metabolism in Streptococcus and its interaction with HPV-16 in the malignant transformation of oral keratinocytes. The acetaldehyde-producing strain Streptococcus gordonii V2016 was analyzed for adh genes and activities of alcohol and aldehyde dehydrogenases. Streptococcus attachment to immortalized HPV-16 infected human oral keratinocytes, HOK (HPV/HOK-16B), human oral buccal keratinocytes, and foreskin keratinocytes was studied. Acetaldehyde, malondialdehyde, DNA damage, and abnormal proliferation among keratinocytes were also quantified. We found 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. S. gordonii V2016 did not show a detectable aldehyde dehydrogenase. AdhE is the major alcohol dehydrogenase in S. gordonii. Acetaldehyde and malondialdehyde production from permissible Streptococcus species significantly increased the bacterial attachment to keratinocytes, which was associated with an enhanced expression of furin to facilitate HPV infection and several malignant phenotypes including acetaldehyde adduct formation, abnormal proliferation, and enhanced migration through integrin-coated basement membrane by HPV-infected oral keratinocytes. Therefore, expression of multiple alcohol dehydrogenases with no functional aldehyde dehydrogenase contributes to excessive production of acetaldehyde from ethanol by oral streptococci. Oral Streptococcus species and HPV may cooperate to transform oral keratinocytes after ethanol exposure. These results suggest a significant clinical interaction, but further validation is warranted.

  11. Isolation and Identification of Genes Activating Uas2-Dependent Adh2 Expression in Saccharomyces Cerevisiae

    PubMed Central

    Donoviel, M. S.; Young, E. T.

    1996-01-01

    Two cis-acting elements have been identified that act synergistically to regulate expression of the glucose-repressed alcohol dehydrogenase 2 (ADH2) gene. UAS1 is bound by the trans-activator Adr1p. UAS2 is thought to be the binding site for an unidentified regulatory protein. A genetic selection based on a UAS2-dependent ADH2 reporter was devised to isolate genes capable of activating UAS2-dependent transcription. One set of UAS2-dependent genes contained SPT6/CRE2/SSN20. Multicopy SPT6 caused improper expression of chromosomal ADH2. A second set of UAS2-dependent clones contained a previously uncharacterized open reading frame designated MEU1 (Multicopy Enhancer of UAS2). A frame shift mutation in MEU1 abolished its ability to activate UAS2-dependent gene expression. Multicopy MEU1 expression suppressed the constitutive ADH2 expression caused by cre2-1. Disruption of MEU1 reduced endogenous ADH2 expression about twofold but had no effect on cell viability or growth. No homologues of MEU1 were identified by low-stringency Southern hybridization of yeast genomic DNA, and no significant homologues were found in the sequence data bases. A MEU1/β-gal fusion protein was not localized to a particular region of the cell. MEU1 is linked to PPR1 on chromosome XII. PMID:8807288

  12. Compensatory evolution of a precursor messenger RNA secondary structure in the Drosophila melanogaster Adh gene

    PubMed Central

    Chen, Ying; Stephan, Wolfgang

    2003-01-01

    Evidence for the evolutionary maintenance of a hairpin structure possibly involved in intron processing had been found in intron 1 of the alcohol dehydrogenase gene (Adh) in diverse Drosophila species. In this study, the putative hairpin structure was evaluated systematically in Drosophila melanogaster by elimination of either side of the stem using site-directed mutagenesis. The effects of these mutations and the compensatory double mutant on intron splicing efficiency and ADH protein production were assayed in Drosophila melanogaster Schneider L2 cells and germ-line transformed adult flies. Mutations that disrupt the putative hairpin structure right upstream of the intron branch point were found to cause a significant reduction in both splicing efficiency and ADH protein production. In contrast, the compensatory double mutant that restores the putative hairpin structure was indistinguishable from the WT in both splicing efficiency and ADH level. It was also observed by mutational analysis that a more stable secondary structure (with a longer stem) in this intron decreases both splicing efficiency and ADH protein production. Implications for RNA secondary structure and intron evolution are discussed. PMID:12972637

  13. 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.

  14. Copy number lability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium).

    PubMed Central

    Small, R L; Wendel, J F

    2000-01-01

    Nuclear-encoded genes exist in families of various sizes. To further our understanding of the evolutionary dynamics of nuclear gene families we present a characterization of the structure and evolution of the alcohol dehydrogenase (Adh) gene family in diploid and tetraploid members of the cotton genus (Gossypium, Malvaceae). A PCR-based approach was employed to isolate and sequence multiple Adh gene family members, and Southern hybridization analyses were used to document variation in gene copy number. Adh gene copy number varies among Gossypium species, with diploids containing at least seven Adh loci in two primary gene lineages. Allotetraploid Gossypium species are inferred to contain at least 14 loci. Intron lengths vary markedly between loci, and one locus has lost two introns usually found in other plant Adh genes. Multiple examples of apparent gene duplication events were observed and at least one case of pseudogenization and one case of gene elimination were also found. Thus, Adh gene family structure is dynamic within this single plant genus. Evolutionary rate estimates differ between loci and in some cases between organismal lineages at the same locus. We suggest that dynamic fluctuation in copy number will prove common for nuclear genes, and we discuss the implications of this perspective for inferences of orthology and functional evolution. PMID:10924485

  15. Redundant cis-acting elements control expression of the Drosophila affinidisjuncta Adh gene in the larval fat body.

    PubMed Central

    McKenzie, R W; Hu, J; Brennan, M D

    1994-01-01

    The alcohol dehydrogenase (Adh) gene in the Hawaiian species of fruit fly, Drosophila affinidisjuncta, like the Adh genes from all Drosophila species analyzed, is expressed at high levels in the larval fat body via a larval-specific promoter. To identify the cis-acting elements involved in this highly conserved aspect of Adh gene expression, deleted D. affinidisjuncta genes were introduced into D. melanogaster by somatic transformation. Unlike previously described methods, this transformation system allows analysis of Adh gene expression specifically in the larval fat body. The arrangement of sequences influencing expression of the proximal promoter of this gene in the larval fat body differs markedly from that described for the Adh gene from the distant relative, D. melanogaster. Multiple redundant elements dispersed 5' and 3' to the gene, only some of which map to regions carrying evolutionarily conserved sequences, affect expression in the fat body. D. affinidisjuncta employs a novel mode of Adh gene regulation in which the proximal promoter is influenced by sequences having roles in expression of the distal promoter. This gene is also unique in that far upstream sequences can compensate for loss of sequences within 200 bp of the proximal RNA start site. Furthermore, expression is influenced in an unusual, context-dependent manner by a naturally-occurring 3' duplication of the proximal promoter--a feature found only in Hawaiian species. Images PMID:8165141

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Screening of Two ADH4 Variations in a Swedish Cluster Headache Case–Control Material

    PubMed Central

    Fourier, Carmen; Ran, Caroline; Steinberg, Anna; Sjöstrand, Christina; Waldenlind, Elisabet

    2016-01-01

    Background Cluster headache (CH) is a severe neurovascular disorder and an increasing amount of evidence points to a genetic contribution to this disease. When CH was first described, it was observed that alcohol may precipitate an attack during the active phase of the disease. The alcohol dehydrogenase 4 (ADH4) gene encodes an enzyme which contributes to the metabolization of alcohol and is, therefore, an interesting candidate gene for CH. Two Italian groups have reported association of the single nucleotide polymorphism (SNP) rs1126671 located in the ADH4 gene with an increased risk of CH in Italy. In addition, one of the groups found an association between the ADH4 SNP rs1800759 and CH. Objective To perform a replication study on the ADH4 SNPs rs1126671 and rs1800759 in a large homogeneous Swedish case–control cohort in order to further investigate the possible contribution of ADH4 to CH. Methods A total of 390 unrelated patients diagnosed with CH and 389 controls representing a general Swedish population were recruited to the study. DNA samples from patients and controls were genotyped for the two ADH4 SNPs rs1126671 and rs1800759 using quantitative real‐time polymerase chain reaction. Statistical analyses of genotype, allele and haplotype frequencies for the two SNPs were performed and compared between patients and controls. Results For rs1126671, the minor allele frequency (A allele) was 32.8% (n = 254) in controls compared with 31.9% (n = 249) in CH patients. The minor allele frequency (A allele) of rs1800759 was 42.3% (n = 324) in controls and 41.9% (n = 327) in CH patients. Statistical analysis showed no significant differences in allele as well as in genotype or haplotype frequencies between the patient and control group for either SNP. This was also seen after stratifying the patient group for experiencing alcohol as a trigger factor. Conclusions The data did not support an association of the ADH4 SNPs rs1126671 and rs1800759 with CH

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse.

    PubMed

    Haseba, Takeshi; Kameyama, Kouji; Mashimo, Keiko; Ohno, Youkichi

    2012-01-01

    ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CL(T) (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its V(max⁡)/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3.

  12. Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse

    PubMed Central

    Haseba, Takeshi; Kameyama, Kouji; Mashimo, Keiko; Ohno, Youkichi

    2012-01-01

    ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CLT (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its Vmax⁡/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3. PMID:22164338

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

    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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. What's My Substrate? Computational Function Assignment of Candida parapsilosis ADH5 by Genome Database Search, Virtual Screening, and QM/MM Calculations.

    PubMed

    Dhoke, Gaurao V; Ensari, Yunus; Davari, Mehdi D; Ruff, Anna Joëlle; Schwaneberg, Ulrich; Bocola, Marco

    2016-07-25

    Zinc-dependent medium chain reductase from Candida parapsilosis can be used in the reduction of carbonyl compounds to pharmacologically important chiral secondary alcohols. To date, the nomenclature of cpADH5 is differing (CPCR2/RCR/SADH) in the literature, and its natural substrate is not known. In this study, we utilized a substrate docking based virtual screening method combined with KEGG, MetaCyc pathway, and Candida genome databases search for the discovery of natural substrates of cpADH5. The virtual screening of 7834 carbonyl compounds from the ZINC database provided 94 aldehydes or methyl/ethyl ketones as putative carbonyl substrates. Out of which, 52 carbonyl substrates of cpADH5 with catalytically active docking pose were identified by employing mechanism based substrate docking protocol. Comparison of the virtual screening results with KEGG, MetaCyc database search, and Candida genome pathway analysis suggest that cpADH5 might be involved in the Ehrlich pathway (reduction of fusel aldehydes in leucine, isoleucine, and valine degradation). Our QM/MM calculations and experimental activity measurements affirmed that butyraldehyde substrates are the potential natural substrates of cpADH5, suggesting a carbonyl reductase role for this enzyme in butyraldehyde reduction in aliphatic amino acid degradation pathways. Phylogenetic tree analysis of known ADHs from Candida albicans shows that cpADH5 is close to caADH5. We therefore propose, according to the experimental substrate identification and sequence similarity, the common name butyraldehyde dehydrogenase cpADH5 for Candida parapsilosis CPCR2/RCR/SADH. PMID:27387009

  2. Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector

    PubMed Central

    Ye, Wei; Zhang, Weimin; Liu, Taomei; Tan, Guohui; Li, Haohua; Huang, Zilei

    2016-01-01

    Bioethanol is becoming increasingly important in energy supply and economic development. However, the low yield of bioethanol and the insufficiency of high-efficient genetic manipulation approaches limit its application. In this study, a novel transcription activator-like effector nuclease (TALEN) vector containing the left and right arms of TALEN was electroporated into Saccharomyces cerevisiae strain As2.4 to sequence the alcohol dehydrogenase gene ADH2 and the hygromycin-resistant gene hyg. Western blot analysis using anti-FLAG monoclonal antibody proved the successful expression of TALE proteins in As2.4 strains. qPCR and sequencing demonstrated the accurate knockout of the 17 bp target gene with 80% efficiency. The TALEN vector and ADH2 PCR product were electroporated into ΔADH2 to complement the ADH2 gene (ADH2+ As2.4). LC–MS and GC were employed to detect ethanol yields in the native As2.4, ΔADH2 As2.4, and ADH2+ As2.4 strains. Results showed that ethanol production was improved by 52.4 ± 5.3% through the disruption of ADH2 in As2.4. The bioethanol yield of ADH2+ As2.4 was nearly the same as that of native As2.4. This study is the first to report on the disruption of a target gene in S. cerevisiae by employing Fast TALEN technology to improve bioethanol yield. This work provides a novel approach for the disruption of a target gene in S. cerevisiae with high efficiency and specificity, thereby promoting the improvement of bioethanol production in S. cerevisiae by metabolic engineering. PMID:27462304

  3. Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector.

    PubMed

    Ye, Wei; Zhang, Weimin; Liu, Taomei; Tan, Guohui; Li, Haohua; Huang, Zilei

    2016-01-01

    Bioethanol is becoming increasingly important in energy supply and economic development. However, the low yield of bioethanol and the insufficiency of high-efficient genetic manipulation approaches limit its application. In this study, a novel transcription activator-like effector nuclease (TALEN) vector containing the left and right arms of TALEN was electroporated into Saccharomyces cerevisiae strain As2.4 to sequence the alcohol dehydrogenase gene ADH2 and the hygromycin-resistant gene hyg. Western blot analysis using anti-FLAG monoclonal antibody proved the successful expression of TALE proteins in As2.4 strains. qPCR and sequencing demonstrated the accurate knockout of the 17 bp target gene with 80% efficiency. The TALEN vector and ADH2 PCR product were electroporated into ΔADH2 to complement the ADH2 gene (ADH2 (+) As2.4). LC-MS and GC were employed to detect ethanol yields in the native As2.4, ΔADH2 As2.4, and ADH2 (+) As2.4 strains. Results showed that ethanol production was improved by 52.4 ± 5.3% through the disruption of ADH2 in As2.4. The bioethanol yield of ADH2 (+) As2.4 was nearly the same as that of native As2.4. This study is the first to report on the disruption of a target gene in S. cerevisiae by employing Fast TALEN technology to improve bioethanol yield. This work provides a novel approach for the disruption of a target gene in S. cerevisiae with high efficiency and specificity, thereby promoting the improvement of bioethanol production in S. cerevisiae by metabolic engineering. PMID:27462304

  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. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Minisatellite instability at the Adh locus reveals somatic polymorphism in amphioxus

    PubMed Central

    Cañestro, Cristian; Gonzàlez-Duarte, Roser; Albalat, Ricard

    2002-01-01

    Amphioxus (subphylum Cephalochordata) is the closest living relative to vertebrates and widely used for phylogenetic analyses of vertebrate gene evolution. Amphioxus genes are highly polymorphic, but the origin and nature of this variability is unknown. We have analyzed the alcohol dehydrogenase locus (Adh3) in two amphioxus species (Branchiostoma lanceolatum and Branchiostoma floridae) and found that genetic variation is related to repetitive DNA sequences, mainly minisatellites. Small pool-PCR assays indicated that allelic variants are generated by minisatellite instability. We conclude that the generation of new forms was not preferentially linked to germline processes but rather to somatic events leading to mosaic adult animals. Furthermore, most Adh minisatellites belong to a novel class, which we have named mirages. Their distinctive feature is that the repeat subunit spans the exon–intron boundaries and generates potential duplications of the splice sites. However, splicing may not be compromised as no aberrant mRNA variants were detected. PMID:12087171

  11. Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol.

    PubMed

    Rauter, Marion; Prokoph, Alexandra; Kasprzak, Jakub; Becker, Karin; Baronian, Keith; Bode, Rüdiger; Kunze, Gotthard; Vorbrodt, H- Matthias

    2015-06-01

    The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200 U l(-1) ADH and 370 U l(-1) G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l(-1) ADH and 170 U l(-1) GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH-LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP(+), makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74 g l(-1) day(-1) or 406 g kg(-1) dry cell weight (dcw) day(-1) isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol product per mole NADP(+). In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.

  12. 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

  13. 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

  14. The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.

    PubMed

    Ogueta, Maite; Cibik, Osman; Eltrop, Rouven; Schneider, Andrea; Scholz, Henrike

    2010-11-01

    Preference determines behavioral choices such as choosing among food sources and mates. One preference-affecting chemical is ethanol, which guides insects to fermenting fruits or leaves. Here, we show that adult Drosophila melanogaster prefer food containing up to 5% ethanol over food without ethanol and avoid food with high levels (23%) of ethanol. Although female and male flies behaved differently at ethanol-containing food sources, there was no sexual dimorphism in the preference for food containing modest ethanol levels. We also investigated whether Drosophila preference, sensitivity and tolerance to ethanol was related to the activity of alcohol dehydrogenase (Adh), the primary ethanol-metabolizing enzyme in D. melanogaster. Impaired Adh function reduced ethanol preference in both D. melanogaster and a related species, D. sechellia. Adh-impaired flies also displayed reduced aversion to high ethanol concentrations, increased sensitivity to the effects of ethanol on postural control, and negative tolerance/sensitization (i.e., a reduction of the increased resistance to ethanol's effects that normally occurs upon repeated exposure). These data strongly indicate a linkage between ethanol-induced behavior and ethanol metabolism in adult fruit flies: Adh deficiency resulted in reduced preference to low ethanol concentrations and reduced aversion to high ones, despite recovery from ethanol being strongly impaired. PMID:20739429

  15. The two small introns of the Drosophila affinidisjuncta Adh gene are required for normal transcription.

    PubMed Central

    McKenzie, R W; Brennan, M D

    1996-01-01

    All Drosophila alcohol dehydrogenase (Adh) genes sequenced to date contain two small introns within the coding region. These are conserved in location and, to some extent, in sequence between the various species analyzed. To determine if these introns play a role in Adh gene expression, derivatives of the Drosophila affinidisjuncta Adh gene lacking one or both introns were constructed and analyzed by germline and transient transformation of Drosophila melanogaster. Removal of both introns lowered expression, whether measured by enzyme activity or by RNA levels. The decrease was seen in both germline transformed and transiently transformed larvae, with the effect being larger for germline transformants. Similar decreases (averaging 5-fold) were also seen at the embryonic and adult stages for germline transformants. Nuclear run-off transcription with nuclei from germline transformed embryos indicated that the reduction in RNA levels is due to decreased transcription. However, LacZ fusion constructs designed to test for the presence of a classical enhancer in the introns provided no evidence for such a mechanism. Removal of each intron individually resulted in more complex phenotypes. The introns have smaller, additive effects on expression in adults. In larvae, removal of the upstream intron significantly increases RNA levels but modestly decreases enzyme activity. Removal of the downstream intron lowers expression in both germline and transiently transformed larvae, but also increases position effects in germline transformants. Therefore, the small introns are clearly needed for optimal transcription of this Adh gene, but multiple mechanisms are involved. PMID:8836194

  16. The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.

    PubMed

    Ogueta, Maite; Cibik, Osman; Eltrop, Rouven; Schneider, Andrea; Scholz, Henrike

    2010-11-01

    Preference determines behavioral choices such as choosing among food sources and mates. One preference-affecting chemical is ethanol, which guides insects to fermenting fruits or leaves. Here, we show that adult Drosophila melanogaster prefer food containing up to 5% ethanol over food without ethanol and avoid food with high levels (23%) of ethanol. Although female and male flies behaved differently at ethanol-containing food sources, there was no sexual dimorphism in the preference for food containing modest ethanol levels. We also investigated whether Drosophila preference, sensitivity and tolerance to ethanol was related to the activity of alcohol dehydrogenase (Adh), the primary ethanol-metabolizing enzyme in D. melanogaster. Impaired Adh function reduced ethanol preference in both D. melanogaster and a related species, D. sechellia. Adh-impaired flies also displayed reduced aversion to high ethanol concentrations, increased sensitivity to the effects of ethanol on postural control, and negative tolerance/sensitization (i.e., a reduction of the increased resistance to ethanol's effects that normally occurs upon repeated exposure). These data strongly indicate a linkage between ethanol-induced behavior and ethanol metabolism in adult fruit flies: Adh deficiency resulted in reduced preference to low ethanol concentrations and reduced aversion to high ones, despite recovery from ethanol being strongly impaired.

  17. A highly efficient ADH-coupled NADH-recycling system for the asymmetric bioreduction of carbon-carbon double bonds using enoate reductases.

    PubMed

    Tauber, Katharina; Hall, Melanie; Kroutil, Wolfgang; Fabian, Walter M F; Faber, Kurt; Glueck, Silvia M

    2011-06-01

    The asymmetric bioreduction of activated alkenes catalyzed by flavin-dependent enoate reductases from the OYE-family represents a powerful method for the production of optically active compounds. For its preparative-scale application, efficient and economic NADH-recycling is crucial. A novel enzyme-coupled NADH-recycling system is proposed based on the concurrent oxidation of a sacrificial sec-alcohol catalyzed by an alcohol dehydrogenase (ADH-A). Due to the highly favorable position of the equilibrium of ene-reduction versus alcohol-oxidation, the cosubstrate is only required in slight excess.

  18. Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco

    PubMed Central

    Ellis, J.G.; Llewellyn, D.J.; Dennis, E.S.; Peacock, W.J.

    1987-01-01

    The promoter region of a maize alcohol dehydrogenase gene (Adh-1) was linked to a reporter gene encoding chloramphenicol acetyl transferase (CAT) and transformed stably into tobacco cells using T-DNA vectors. No CAT enzyme activity could be detected in transgenic tobacco plants unless upstream promoter elements from the octopine synthase gene or the cauliflower mosaic virus 35S promoter were supplied in addition to the maize promoter region. CAT enzyme activity and transcription of the chimaeric gene were then readily detected after anaerobic induction. The first 247 bp upstream of the translation initiation codon of the maize Adh-1 gene were sufficient to impose anaerobic regulation on the hybrid gene and S1 nuclease mapping confirmed mRNA initiation is from the normal maize Adh-1 transcription start point. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:15981329

  19. Maternal Alcohol Consumption, Alcohol Metabolism Genes, and the Risk of Oral Clefts: A Population-based Case-Control Study in Norway, 1996–2001

    PubMed Central

    Boyles, Abee L.; DeRoo, Lisa A.; Lie, Rolv T.; Taylor, Jack A.; Jugessur, Astanand; Murray, Jeffrey C.; Wilcox, Allen J.

    2010-01-01

    Heavy maternal alcohol consumption during early pregnancy increases the risk of oral clefts, but little is known about how genetic variation in alcohol metabolism affects this association. Variants in the alcohol dehydrogenase 1C (ADH1C) gene may modify the association between alcohol and clefts. In a population-based case-control study carried out in Norway (1996–2001), the authors examined the association between maternal alcohol consumption and risk of oral clefts according to mother and infant ADH1C haplotypes encoding fast or slow alcohol-metabolizing phenotypes. Subjects were 483 infants with oral cleft malformations and 503 control infants and their mothers, randomly selected from all other livebirths taking place during the same period. Mothers who consumed 5 or more alcoholic drinks per sitting during the first trimester of pregnancy had an elevated risk of oral cleft in their offspring (odds ratio (OR) = 2.6, 95% confidence interval (CI): 1.4, 4.7). This increased risk was evident only in mothers or children who carried the ADH1C haplotype associated with reduced alcohol metabolism (OR= 3.0, 95% CI: 1.4, 6.8). There was no evidence of alcohol-related risk when both mother and infant carried only the rapid-metabolism ADH1C variant (OR = 0.9, 95% CI: 0.2, 4.1). The teratogenic effect of alcohol may depend on the genetic capacity of the mother and fetus to metabolize alcohol. PMID:20810466

  20. 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

  1. Mechanisms of mutagenesis: analysis through the use of alcohol dehydrogenase in Drosophila. Progress report, February 1984-January 1985

    SciTech Connect

    Sofer, W.H.

    1985-01-01

    A technique was developed for somatic transformation, making it possible to inject embryos with ADH genes in a variety of forms. A selection procedure was developed to chemically select strains of flies that are lacking beta - galactosidase activity. Two strains of Adh-negative flies whose lesions were generated with formaldehyde were characterized with respect to the ADH protein they manufacture, the rate at which they synthesize ADH, and the nature of the ADH mNRNA they make. (ACR)

  2. Use of in Vitro Mutagenesis to Analyze the Molecular Basis of the Difference in Adh Expression Associated with the Allozyme Polymorphism in Drosophila Melanogaster

    PubMed Central

    Choudhary, M.; Laurie, C. C.

    1991-01-01

    In natural populations of Drosophila melanogaster, the alcohol dehydrogenase (Adh) locus is polymorphic for two allozymes, designated Slow and Fast. Fast homozygotes generally have a two- to threefold higher ADH activity level than Slow homozygotes for two reasons: they have a higher concentration of ADH protein and the Fast protein has a higher catalytic efficiency. DNA sequencing studies have shown that the two allozymes generally differ by only a single amino acid at residue 192, which must therefore be the cause of the catalytic efficiency difference. A previous P element-transformation experiment mapped the difference in ADH protein level to a 2.3-kb HpaI/ClaI restriction fragment, which contains all of the Adh coding sequences but excludes all of the 5' flanking region of the distal transcriptional unit. Here we report the results of a site-directed in vitro mutagenesis experiment designed to investigate the effects of the amino acid replacement. This replacement has the expected effect on catalytic efficiency, but there is no detectable effect on the concentration of ADH protein estimated immunologically. This result shows that the average difference in ADH protein level between the allozymic classes is due to linkage disequilibrium between the amino acid replacement and one or more other polymorphisms within the HpaI/ClaI fragment. Sequence analysis of several Fast and Slow alleles suggested that the other polymorphism might be a silent substitution at nucleotide 1443, but another in vitro mutagenesis experiment reported here shows that this is not the case. Therefore, the molecular basis of the difference in ADH protein concentration between the allozymic classes remains an open question. PMID:1743488

  3. 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.

  4. Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase

    PubMed Central

    Bae, Sang-Jeong; Kim, Sujin; Hahn, Ji-Sook

    2016-01-01

    Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study, Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes (ADH1, ADH2, ADH3, ADH4, and ADH5) and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD+ regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose. PMID:27279026

  5. Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase.

    PubMed

    Bae, Sang-Jeong; Kim, Sujin; Hahn, Ji-Sook

    2016-01-01

    Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study, Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes (ADH1, ADH2, ADH3, ADH4, and ADH5) and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD(+) regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose. PMID:27279026

  6. 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.

  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. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. Molecular analysis of the Adh region of the genome of Drosophila melanogaster.

    PubMed

    Chia, W; Karp, R; McGill, S; Ashburner, M

    1985-12-20

    A small region of the genome of Drosophila melanogaster has been cloned in a series of overlapping phage. A length of 165 X 10(3) base-pairs of contiguous DNA that spans polytene chromosome region 35A4 to 35B1 and includes the structural gene for alcohol dehydrogenase (Adh) as well as at least two other genes, outspread (osp) and no-ocelli (noc), has been characterized by mapping chromosome aberrations to the DNA. The relationship between osp and Adh is surprising: of nine osp alleles associated with chromosome breakpoints, five map distal (i.e. 5') to Adh and four map proximal (i.e. 3') to this gene. None affects the expression of Adh. As defined by these and other breakpoints, the osp gene spans at least 52 X 10(3) base-pairs and overlaps the Adh gene. The noc gene, as defined by the mapping of nearly 30 breakpoints, is at least 50 X 10(3) base-pairs in size. Alleles of noc and noc- deletions show either of two kinds of interaction with the recessive lethality of l(2)br29ScoR+1, a lethal that maps immediately distal to noc. One class of noc allele is viable when heterozygous with ScoR+1, while the other class is lethal or semi-lethal. Both classes, however, are homozygous or hemizygous viable. The locations of these two classes of noc allele on the DNA fall into two clusters, with those that are viable with ScoR+1 located proximal to those that are not. The physical boundary between these classes lies at a site just distal to that of the breakpoint of the inversion associated with ScoR+1 itself.

  16. 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.

  17. 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.

  18. Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

    PubMed Central

    Gallego, Oriol; Belyaeva, Olga V.; Porté, Sergio; Ruiz, F. Xavier; Stetsenko, Anton V.; Shabrova, Elena V.; Kostereva, Natalia V.; Farrés, Jaume; Parés, Xavier; Kedishvili, Natalia Y.

    2006-01-01

    Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low Km values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low Km values for retinoids (0.12–1.1 μM), whilst they strongly differ in their kcat values, which range from 0.35 min−1 for AKR1B1 to 302 min−1 for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo. PMID:16787387

  19. 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.

  20. 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

  1. Dissociation and rate constants of some human liver alcohol dehydrogenase isoenzymes.

    PubMed

    Pietruszko, R; de Zalenski, C; Theorell, H

    1976-01-01

    ADH from human liver forms binary complexes with NADH, associated with a blue shift of the peak of the fluorescence emission of NADH. The wavelength shift is the same for all isoenzymes but the accompanying intensification of the fluorescence is different. The fluorescence is further increased by the formation of the very tight ternary enzyme-NADH-isobutyramide complexes. These properties are similar to those for the horse liver ADH, as well as the molecular weight of E=40 000 per active site of the dimer molecule (EE). "Stopped-flow" determined velocity constants (ER in equilibrium E+R) were found to be in good agreement with ethanol activity constants previously determined by activity measurement, confirming the validity of the ordered ternary complex mechanism also for the human ADH. No single isoenzyme activity as high as that reported by Mourad and Woronick or Drum has been found. PMID:184631

  2. Co-expression of TAL1 and ADH1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural.

    PubMed

    Hasunuma, Tomohisa; Ismail, Ku Syahidah Ku; Nambu, Yumiko; Kondo, Akihiko

    2014-02-01

    Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production.

  3. Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency.

    PubMed

    Min, Ting; Yin, Xue-ren; Shi, Yan-na; Luo, Zheng-rong; Yao, Yun-cong; Grierson, Donald; Ferguson, Ian B; Chen, Kun-song

    2012-11-01

    The persimmon fruit is a particularly good model for studying fruit response to hypoxia, in particular, the hypoxia-response ERF (HRE) genes. An anaerobic environment reduces fruit astringency by converting soluble condensed tannins (SCTs) into an insoluble form. Although the physiology of de-astringency has been widely studied, its molecular control is poorly understood. Both CO(2) and ethylene treatments efficiently removed the astringency from 'Mopan' persimmon fruit, as indicated by a decrease in SCTs. Acetaldehyde, the putative agent for causing de-astringency, accumulated during these treatments, as did activities of the key enzymes of acetaldehyde synthesis, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC). Eight DkADH and DkPDC genes were isolated, and three candidates for a role in de-astringency, DkADH1, DkPDC1, and DkPDC2, were characterized by transcriptional analysis in different tissues. The significance of these specific isoforms was confirmed by principal component analysis. Transient expression in leaf tissue showed that DkPDC2 decreased SCTs. Interactions of six hypoxia-responsive ERF genes and target promoters were tested in transient assays. The results indicated that two hypoxia-responsive ERF genes, DkERF9 and DkERF10, were involved in separately regulating the DkPDC2 and DkADH1 promoters. It is proposed that a DkERF-DkADH/DkPDC cascade is involved in regulating persimmon de-astringency.

  4. Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency

    PubMed Central

    Min, Ting; Yin, Xue-ren; Chen, Kun-song

    2012-01-01

    The persimmon fruit is a particularly good model for studying fruit response to hypoxia, in particular, the hypoxia-response ERF (HRE) genes. An anaerobic environment reduces fruit astringency by converting soluble condensed tannins (SCTs) into an insoluble form. Although the physiology of de-astringency has been widely studied, its molecular control is poorly understood. Both CO2 and ethylene treatments efficiently removed the astringency from ‘Mopan’ persimmon fruit, as indicated by a decrease in SCTs. Acetaldehyde, the putative agent for causing de-astringency, accumulated during these treatments, as did activities of the key enzymes of acetaldehyde synthesis, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC). Eight DkADH and DkPDC genes were isolated, and three candidates for a role in de-astringency, DkADH1, DkPDC1, and DkPDC2, were characterized by transcriptional analysis in different tissues. The significance of these specific isoforms was confirmed by principal component analysis. Transient expression in leaf tissue showed that DkPDC2 decreased SCTs. Interactions of six hypoxia-responsive ERF genes and target promoters were tested in transient assays. The results indicated that two hypoxia-responsive ERF genes, DkERF9 and DkERF10, were involved in separately regulating the DkPDC2 and DkADH1 promoters. It is proposed that a DkERF–DkADH/DkPDC cascade is involved in regulating persimmon de-astringency. PMID:23095993

  5. Nucleotide sequence analysis of Adh genes estimates the time of geographic isolation of the Bogota population of Drosophila pseudoobscura.

    PubMed Central

    Schaeffer, S W; Miller, E L

    1991-01-01

    The population of Drosophila pseudoobscura at Bogota, Columbia, is geographically and partially reproductively isolated from populations in the main body of the species in North America. The degree of genetic differentiation and time of divergence between populations at Bogota and Apple Hill, CA, were estimated by comparison of 3388 nucleotides in the alcohol dehydrogenase region (Adh and Adh-Dup genes) of 18 strains. Of the 146 polymorphic nucleotide sites detected, 68 and 31 were unique to the Apple Hill and Bogota samples, respectively, and 53 were shared. On the basis of an observed net divergence per nucleotide site of 0.264% between the two samples, the Bogota and North American populations were estimated to have been separated for at least 155,000 years. This divergence time suggests that D. pseudoobscura extended its range from North America to South America in a period of Pleistocene glaciation, when habitat suitable for the species presumably existed in lowland Central America. PMID:2068088

  6. Chaperone-like activity of alpha-cyclodextrin via hydrophobic nanocavity to protect native structure of ADH.

    PubMed

    Barzegar, Abolfazl; Moosavi-Movahedi, Ali A; Mahnam, Karim; Ashtiani, Saman Hosseini

    2010-01-26

    The chaperone action of alpha-cyclodextrin (alpha-CyD), based on providing beneficial microenvironment of hydrophobic nanocavity to form molecular complex with alcohol dehydrogenase (ADH) was examined by experimental and computational techniques. The results of UV-vis and dynamic light scattering (DLS) indicated that the chaperone-like activity of alpha-CyD depends on molecular complex formation between alpha-CyD and ADH, which caused to decrease the amount and size of polymerized molecules. Computational calculations of molecular dynamic (MD) simulations and blind docking (BD) demonstrated that alpha-CyD acts as an artificial chaperone because of its high affinity to the region of ADH's two chains interface. The hydrophobic nanocavity of alpha-CyD has the ability to form inclusion complex due to the presence of phenyl ring of aromatic phenylalanine (Phe) residue in the dimeric intersection area. Delocalization of ADH subunits, which causes the exposure of Phe110, takes part in the enzyme polymerization and has proven to be beneficial for aggregation inhibition and solubility enhancement within the host alpha-CyD-nanocavity.

  7. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1986-03-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. Focus is on the two final steps in alcohol synthesis, which are catalyzed by alcohol dehydrogenase and acetaldehyde CoA dehydrogenase. We have isolated a series of mutations affecting the expression of these enzymes. Some of these mutations are in the structural genes for these enzymes; others affect the regulation of the adh operon. We have recently cloned the genes coding for these enzymes and are now studying the effect of multiple copies of the adh gene on fermentative growth and its regulation. A recently invented technique, proton suicide has allowed the selection of a variety of novel mutants affecting fermentation which are presently being characterized. We have isolated a comprehensive collection of operon fusions in which the lacZ structural gene is fused to promoters that are inactive aerobically but active anaerobically. Although these genes (like adh) are only expressed under anaerobic conditions, the level of induction varies from two-fold to nearly 100-fold. The nitrogen source, medium pH, nature of the buffer, presence of alternative electron acceptors (e.g., nitrate), and other factors exert a great effect on the expression of many of these genes. In the near future we will investigate control mechanisms common to the adh operon and other anaerobically regulated genes.

  8. Effects of methylmercury and alcohol exposure in Drosophila melanogaster: Potential risks in neurodevelopmental disorders.

    PubMed

    Chauhan, Ved; Chauhan, Abha

    2016-06-01

    Extensive evidence suggests the role of oxidative stress in autism and other neurodevelopmental disorders. In this study, we investigated whether methylmercury (MeHg) and/or alcohol exposure has deleterious effects in Drosophila melanogaster (fruit flies). A diet containing different concentrations of MeHg in Drosophila induced free radical generation and increased lipid peroxidation (markers of oxidative stress) in a dose-dependent manner. This effect of MeHg on oxidative stress was enhanced by further exposure to alcohol. It was observed that alcohol alone could also induce free radical generation in flies. After alcohol exposure, MeHg did not affect the immobilization of flies, but it increased the recovery time in a concentration-dependent manner. MeHg significantly inhibited the activity of alcohol dehydrogenase (ADH) in a dose-dependent manner. Linear regression analysis showed a significant negative correlation between ADH activity and recovery time upon alcohol exposure in the flies fed a diet with MeHg. This relationship between ADH activity and recovery time after alcohol exposure was confirmed by adding 4-methyl pyrazole (an inhibitor of ADH) to the diet for the flies. These results suggest that consumption of alcohol by pregnant mothers who are exposed to MeHg may lead to increased oxidative stress and to increased length of time for alcohol clearance, which may have a direct impact on the development of the fetus, thereby increasing the risk of neurodevelopmental disorders. PMID:27151262

  9. 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

  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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Association of genetic polymorphisms in ADH and ALDH2 with risk of coronary artery disease and myocardial infarction: a meta-analysis.

    PubMed

    Han, Hongguang; Wang, Huishan; Yin, Zongtao; Jiang, Hui; Fang, Minhua; Han, Jingsong

    2013-09-10

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the major enzymes responsible for alcohol metabolism in humans. Emerging evidences have shown that functional polymorphisms in ADH and ALDH genes might play a critical role in increasing coronary artery disease (CAD) and myocardial infarction (MI) risks; however, individually published studies showed inconclusive results. The aim of this meta-analysis is to evaluate the associations between the genetic polymorphisms of ADH and ALDH genes with susceptibility to CAD and MI. A literature search was conducted on PubMed, Embase, Web of Science and Chinese BioMedical databases from inception through December 1st, 2012. Crude relative risks (RRs) with 95% confidence intervals (CIs) were calculated. Twelve case-control studies were included with a total of 9616 subjects, including 2053 CAD patients, 1436 MI patients, and 6127 healthy controls. Meta-analysis showed that mutant genotypes (GA+AA) of the rs671 polymorphism in the ALDH2 gene were associated with increased risk of both CAD and MI (CAD: RR=1.20, 95%CI: 1.03-1.40, P=0.021; MI: RR=1.32, 95%CI: 1.11-1.57, P=0.002). However, there were no significant associations of ADH genetic polymorphisms to CAD and MI risks (CAD: RR=0.92, 95%CI: 0.73-1.15, P=0.445; MI: RR=0.93, 95%CI: 0.84-1.03, P=0.148). In conclusion, this meta-analysis provides strong evidence that ALDH2 rs671 polymorphism may be associated with increased risks of CAD and MI. However, further studies are still needed to accurately determine whether ADH genetic polymorphisms are associated with susceptibility to CAD and MI.

  16. Temperature and water loss affect ADH activity and gene expression in grape berry during postharvest dehydration.

    PubMed

    Cirilli, Marco; Bellincontro, Andrea; De Santis, Diana; Botondi, Rinaldo; Colao, Maria Chiara; Muleo, Rosario; Mencarelli, Fabio

    2012-05-01

    Clusters of Aleatico wine grape were picked at 18°Brix and placed at 10, 20, or 30°C, 45% relative humidity (RH) and 1.5m/s of air flow to dehydrate the berries up to 40% of loss of initial fresh weight. Sampling was done at 0%, 10%, 20%, 30%, and 40% weight loss (wl). ADH (alcohol dehydrogenase) gene expression, enzyme activity, and related metabolites were analysed. At 10°C, acetaldehyde increased rapidly and then declined, while ethanol continued to rise. At 20°C, acetaldehyde and ethanol increased significantly with the same pattern and declined at 40%wl. At 30°C, acetaldehyde did not increase but ethanol increased rapidly already at 10%wl. At the latter temperature, a significant increase in acetic acid and ethyl acetate occurred, while at 10°C their values were low. At 30°C, the ADH activity (ethanol to acetaldehyde direction), increased rapidly but acetaldehyde did not rise because of its oxidation to acetic acid, which increased together with ethyl acetate. At 10°C, the ADH activity increased at 20%wl and continued to rise even at 40%wl, meaning that ethanol oxidation was delayed. At 20°C, the behaviour was intermediate to the other temperatures. The relative expression of the VvAdh2 gene was the highest at 10°C already at 10%wl in a synchrony with the ADH activity, indicating a rapid response likely due to low temperature. The expression subsequently declined. At 20 and 30°C, the expression was lower and increased slightly during dehydration in combination with the ADH activity. This imbalance between gene expression and ADH activity at 10°C, as well as the unexpected expression of the carotenoid cleavage dioxygenase 1 (CCD1) gene, opens the discussion on the stress sensitivity and transcription event during postharvest dehydration, and the importance of carefully monitoring temperature during dehydration.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial ch