Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5′-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.     

Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5'-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.     

Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3.

Mammalian alcohol dehydrogenase (ADH) catalyzes the oxidation of retinol to retinaldehyde, the rate-limiting step in the synthesis of retinoic acid. There exists a family of ADH isozymes encoded by unique genes, and it is unclear which isozymes are most important for regulation of retinoic acid synthesis during differentiation or development. A region in the human ADH3 promoter from -328 to -272 base pairs was shown previously to function as a retinoic acid response element (RARE), prompting an hypothesis for a positive feedback mechanism controlling retinoic acid synthesis (Duester, G., Shean, M. L., McBride, M. S., and Stewart, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646). The ADH3 RARE contains three direct AGGTCA repeats which constitute the critical nucleotides of RAREs present in other genes. We dissected the ADH3 RARE and determined that receptor binding as well as transactivation are dependent upon only the two downstream AGGTCA motifs separated by 5 base pairs, a structure noticed previously for a RARE in the promoter for the retinoic acid receptor beta (RAR beta) gene. ADH3 and RAR beta RAREs functioned similarly in transfection assays, suggesting that the feedback mechanisms controlling ADH3 and RAR beta utilize a common RARE. We also found that the normal functioning of the ADH3 RARE was abrogated by thyroid hormone receptor in the presence of thyroid hormone. A negative thyroid hormone response element in the human ADH3 promoter was found to colocalize with the RARE. Since ADH production in rat liver is known to be repressed by thyroid hormone, these findings suggest that human ADH production may also be subject to thyroid hormone repression and that the mechanism involves an interference with retinoic acid induction.     

Inactivation of alcohol dehydrogenase (ADH) by ferryl derivatives of human hemoglobin

In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H2O2 with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H2O2 was added to the mixture of metHb and ADH [the (metHb+ADH)+H2O2] system (ADH was present in the system since the moment of addition of H2O2 i. e. since the very beginning of the reaction of metHb with H2O2). In the second system ADH was added to the system 5 min after the initiation of the reaction of H2O2 with metHb [the (metHb+H2O2)5 min+ADH] system. In the first case all the products of reaction of H2O2 with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV)=O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H2O2 with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H2O2 with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical forms dominated the reaction of inactivation.     

Molecular structure of the human alcohol dehydrogenase 3 gene.

The structure and nucleotide sequence of an ADH3(1) allele, which encodes the ADH gamma 1 subunit, have been determined. The intron positions of the ADH3 gene are identical to those of the other class I and class II ADH genes. The level of nucleotide variation at the ADH3 locus is somewhat higher than those at the ADH1 and ADH2 loci.     

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

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.     

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.     

Polymorphism of alcohol dehydrogenase gene ADH1B in eastern Slavic and Iranian-speaking populations

Frequencies of alleles and genotypes for alcohol dehydrogenase gene ADH1B (arg47his polymorphism), associated with alcohol tolerance/sensitivity, were determined. It was demonstrated that the frequency of allele ADH1B*47his, corresponding to atypical alcohol dehydrogenase variant in Russians, Ukrainians, Iranians, and mountain-dwellers of the Pamirs constituted 3, 7, 24, and 22%, respectively. The frequencies established were consistent with the allele frequency distribution pattern among the populations of Eurasia. Russians and Ukrainians were indistinguishable from other European populations relative to the frequency of allele ADH1B*47his, and consequently, relative to specific features of ethanol metabolic pathways. The data obtained provide refinement of the geographic pattern of ADH1B*47his frequency distribution in Eurasia.     

Multiple mRNAs for human alcohol dehydrogenase (ADH): developmental and tissue specific differences.

Human class I alcohol dehydrogenase (ADH) genes show developmental and tissue specific differences in expression at the polypeptide level. In these studies ADH expression was investigated at the RNA level. Northern blot analysis of total and poly (A) RNA from adult liver using pADH12 probe demonstrated multiple RNA size classes of 2.6, 2.2, 1.9 and 1.6kb. In contrast, fetal liver, and fetal intestine contained only 2.6 and 1.6kb mRNA while fetal lung showed only 2.6kb mRNA. All of these tissues showed a relative reduction in the amount of ADH mRNA present when compared to adult liver. Immunoprecipitation of in vitro translation products of adult liver RNA by polyclonal ADH antibody revealed a single polypeptide of 40,000 daltons. This result points out the homogeneity of size of class I ADH polypeptides despite mRNA size diversity. Variation in length of the 3' untranslated region probably contributes to the multiple size classes of ADH mRNA observed.     

Molecular cloning and characterization of the alcohol dehydrogenase ADH1 gene of Candida utilis ATCC 9950

The alcohol dehydrogenase gene (ADH1) of Candida utilis ATCC9950 was cloned and expressed in recombinant Escherichia coli. C. utilis ADH1 was obtained by PCR amplification of C. utilis genomic DNA using two degenerate primers. Amino acid sequence analysis of C. utilis ADH1 indicated that it contained a zinc-binding consensus region and a NAD(P)⁺-binding site, and lacked a mitochondrial targeting peptide. It has a 98 and 73% identity with ADH1s of C. albicans and Saccharomyces cerevisiae, respectively. Amino acid sequence analysis and enzyme characterization with various aliphatic and branched alcohols suggested that C. utilis ADH1 might be a primary alcohol dehydrogenase existing in the cytoplasm and requiring zinc ion and NAD(P)⁺ for reaction.     

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

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.