Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5

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.     

Multiplication of the class I alcohol dehydrogenase locus in mammalian evolution

Chromosomal DNA samples derived from various primates and other mammals (horse, sheep, rabbit, and mouse) were digested with restriction endonuclease and hybridized with a probe of the sixth exon of the human ADH gene, which is highly conserved in the class I alcohol dehydrogenase of these mammalian species. The copy number of the class I ADH gene in each species was estimated from the number of hybridized bands. Primate DNA samples showed three distinct bands in the blots of PstI digest and DraI digest. Moreover, most of the bands from primate DNA showed a similarity in size so as to allow us to assign the ADH1, ADH2, and ADH3 homologues in each species. In contrast, mouse has only one gene, and rabbit, sheep, and horse seem to have only two genes, for the class I ADH, which showed divergent hybridization bands. These results are consistent with the view that the human class I ADH gene cluster has been generated through gene multiplication events which occurred before the Catarrhini branch point in the course of primate evolution.     

Oxidation of thiodiglycol (2,2'-thiobis-ethanol) by alcohol dehydrogenase: comparison of human isoenzymes

Sulfur mustard is a chemical warfare agent that causes blistering of the skin and damages the eyes and airway after environmental exposure. We have previously reported that thiodiglycol (TDG, 2,2'-bis-thiodiethanol), the hydrolysis product of sulfur mustard, is oxidized by alcohol dehydrogenase (ADH) purified from horse liver or present in mouse liver and human skin cytosol. Humans express four functional classes of ADH composed of several different isozymes, which vary in their tissue distribution, some occurring in skin. To help us evaluate the potential contribution of the various human isozymes toward toxicity in skin and in other tissues, we have compared the catalytic activity of purified human class I alphaalpha-, beta1beta1-, beta2beta2-, and gamma1gamma1-ADH, class II pi-ADH, class III chi-ADH, and class IV sigma-ADH with respect to TDG oxidation and their relative sensitivities to inhibition by pyrazole. Specific activities toward TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the class I alphaalpha-, beta1,beta1-, beta2beta2-, and gamma1gamma1-ADH and class II pi-ADH, respectively. TDG was not a substrate for class III chi-ADH. The specific activity of class IV sigma-ADH was estimated at about 1630 nmol/min/mg. 1 mM pyrazole, a potent inhibitor of class I ADH, inhibited the class I alphaalpha, beta1beta1, beta2beta2, and gamma1gamma1 ADH and class IV sigma-ADH by 83, 100, 56, 90, and 73%, respectively. The class I alphaalpha- and beta1beta1-ADH oxidized TDG with kcat/Km value of 7-8 mM(-1) min(-1), beta2beta2-ADH with a value 19 mM(-1) min(-1) and class I gamma1gamma1-ADH with a value of 176 mM(-1) min(-1). The kcat/Km value for class IV sigma-ADH was estimated at 4 mM(-1) min(-1). The activities of class IV sigma-ADH and class I gamma1gamma1-ADH are of significant interest because of their prevalence in eyes, lungs, stomach, and skin, all target organs of sulfur mustard toxicity.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

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.     

Mammalian alcohol dehydrogenase — Functional and structural implications

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1–ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.     

Differences in charge and kinetic properties of alcohol dehydrogenase 4 from C57BL/6 mice compared to other inbred strains are associated with a cysteine120 to arginine120 substitution

Alcohol dehydrogenase class IV (ADH4) participates in retinol metabolism and is expressed primarily in ocular, digestive, and reproductive tissues of the mouse. A naturally occurring genetic variant in C57BL/6J mice results in a faster migrating ADH4 enzyme during electrophoresis when compared to other non-C57BJ/6J strains. The C57BL/6 ADH4 gene coding sequence is found to have two nucleotide substitutions when compared to the gene from C3HeB/FeJ mice. The substitution in exon 5 encodes Arg120 instead of Cys120 in C57BL/6 ADH4 polypeptide; that would account for the protein electrophoretic phenotype. Arg120 is present in all published mammalian ADH4 sequences but is only in a limited number of mouse strains. The Arg120 residue is part of the outer loop of the substrate binding pocket and appears to have an effect on the affinity of the enzyme for several substrates.     

Linkage disequilibrium at the ADH2 and ADH3 loci and risk of alcoholism

Two of the three class I alcohol dehydrogenase (ADH) genes (ADH2 and ADH3) encode known functional variants that act on alcohol with different efficiencies. Variants at both these genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) we found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2. The class I ADH genes all lie within 80 kb on chromosome 4; thus, variants are not inherited independently, and haplotypes must be analyzed when evaluating the risk of alcoholism. In the Taiwanese Chinese we found that, only among those chromosomes containing the ADH3*1 variant (high Vmax), the proportions of chromosomes with ADH2*1 (low Vmax) and those with ADH2*2 (high Vmax) are significantly different between alcoholics and controls (P<10-5). The proportions of chromosomes with ADH3*1 and those with ADH3*2 are not significantly different between alcoholics and controls, on a constant ADH2 background (with ADH2*1, P=.83; with ADH2*2, P=.53). Thus, the observed differences in the frequency of the functional polymorphism at ADH3, between alcoholics and controls, can be accounted for by the disequilibrium with ADH2 in this population.     

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.     

Drosophila fat body protein P6 and alcohol dehydrogenase are derived from a common ancestral protein

Summary Drosophila melanogaster alcohol dehydrogenase is an example of convergent evolution: it is not related to the ADHs of other organisms, but to short-chain dehydrogenases, which until now have been found only in bacteria and in mammalian steroid hormone metabolism. We present evidence that theDrosophila ADH is phylogenetically more closely related to P6, another highly expressed protein from the fat body ofDrosophila, than it is to the short-chain dehydrogenases. The polypeptide sequence of P6 was inferred from DNA sequence analysis. Both ADH and P6 polypeptides have retained a high structural similarity with respect to the Chou-Fasman prediction of secondary structure and hydropathy. P6 is also homologous to the 25-kd protein from the fat body ofSarcophaga peregrina, whose sequence we have reexamined. The evolution of the P6-ADH family of proteins is characterized by a dramatic increase in the methionine content of P6. Methionine accounts for 20% of P6 amino acids. This is in contrast with the absence of this amino acid in mature ADH. There is evidence that P6 and the 25-kd protein have undergone a parallel and independent enrichment in methionine. When corrected for this, the rate of amino acid replacement shows that the P6-25-kd lineage diverged from insect ADH shortly before the divergence of the ADH gene (Adh) from its 3-duplication (Adhdup).     

Isolation and characterization of shrew (Suncus murinus) liver alcohol dehydrogenases

1. Starch gel electrophoresis of adult shrew (Suncus murinus) liver extracts revealed five forms of alcohol dehydrogenase (ADH 1-5) and four of them were purified. 2. ADH-4 and ADH-5 resemble human class I ADH in terms of electrophoretic mobility, substrate specificity and sensitivity to pyrazole inhibition. 3. ADH-2 does not belong to any of the three classes of human ADHs but rather with catalytic properties similar to those of the class B ADH found in guinea pig liver. 4. ADH-1 prefers secondary alcohol over primary alcohol substrates and between the enantiomers tested, the enzyme favors the S isomers.     

A human liver alcohol dehydrogenase enzyme-linked immunosorbent assay method specific for class I, II, and III isozymes

A sensitive and convenient method for the quantitative measurement of human alcohol dehydrogenase (ADH) isozymes based on enzyme-linked immunosorbent assay has been devised. The procedure was optimized with respect to antigen coating density, antiserum dilution, and incubation times with rabbit antisera raised against beta 1 beta 1-ADH to achieve a limit of sensitivity of 1 ng/ml for this isozyme when purified. Using the optimal conditions established, quantitative measurement of alpha beta 1, alpha gamma 1, beta 1 gamma 1, pi, and chi-ADH were obtained with antisera raised in rabbits toward these individual isozymes. The incorporation into the procedure of thimerosal (ethyl(4-mercaptobenzoato-S)mercury) or other sulfhydryl specific reagents improved the soluble phase antiserum avidity for all ADH isozymes, thereby increasing the sensitivity. Thimerosal is an absolute requirement for chi-ADH antigen-antibody binding. The polyclonal rabbit antisera elicited by the individual isozymes of the three classes of ADH exhibit a high degree of isozyme class specificity. Cross-reactivity of the antibodies with the beta 1 beta 1, alpha gamma 1, alpha gamma 2, alpha beta 1, beta 1 gamma 1, beta 1 gamma 2, pi and chi isozymes were evaluated. Antisera against the class I isozymes beta 1 beta 1 and beta 1 gamma 1 cross-react with all class I isozymes and with pi-ADH. Antibodies against pi and chi-ADH are selective and specific only for their respective antigens. Neither one cross-reacts with any class I isozyme. Conformational effects resulting from subunit interactions likely account for differences in cross-immunoreactivity between the closely homologous class I isozymes.     

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio)

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.     

Alcohol dehydrogenase genes: restriction fragment length polymorphisms for ADH4 (π-ADH) and ADH5 (χ-ADH) and construction of haplotypes among different ADH classes

Of the five human alcohol dehydrogenase (ADH) genes located in the region q21–25 of chromosome 4, genetic markers have been reported previously only for class I enzymes, ADH1-3. Here, new restriction fragment length polymorphisms (RFLPs) are described for the genes of two other classes, ADH4 () and ADH5 ( or formaldehyde dehydrogenase, FDH). The frequencies and modes of inheritance of these RFLPs were determined with DNA both from unrelated individuals and from families. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. Pairwise linkage disequilibrium calculations for these new RFLPs and already known RFLPs at the ADH2 and ADH3 loci establish strong linkage disequilibria between polymorphic MspI and BstXI sites in the ADH5 gene as well as between XbaI and MspI sites in the ADH3 gene. Furthermore, linkage disequilibria were detected between RFLPs of the ADH2 and ADH3 genes as well as between those of the ADH4 and ADH5 genes. The latter disequilibrium implies a hitherto unknown physical proximity of two genes belonging to different ADH classes. The RFLPs were used to construct chromosomal haplotypes that include three ADH classes. Of the 16 possible haplotypes for four RFLP markers used here, 10 were experimentally detected. The potential application of the ADH RFLPs and haplotypes in linkage or association studies of inherited diseases such as familial alcoholism is discussed.