The major piscine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III.

The major alcohol dehydrogenase of cod liver has been purified, enzymatically characterized, and structurally analyzed in order to establish original functions and relationships among the deviating classes of the enzyme in mammalian tissues. Interestingly, the cod enzyme exhibits mixed properties--many positional identities with a class III protein, but functionally a class I enzyme--blurring the distinction among the classes of alcohol dehydrogenase. The two domain interfaces, affected by movements upon coenzyme binding, both exhibit substitutions in a manner thus far unique to the cod enzyme. In contrast, coenzyme-binding residues are highly conserved. At the active site, inner and outer parts of the substrate pocket show different extents of amino acid replacement. In total, no less than 7-10 residues of 11 in the substrate binding pocket differ from those of all the mammalian classes, explaining the substrate specificities. However, the inner part of the substrate pocket is very similar to that of the class I enzymes, which is compatible with the observed characteristics of the cod enzyme: ethanol is an excellent substrate (Km = 1.2 mM) and 4-methylpyrazole is a strong inhibitor (Ki = 0.1 microM). These values are about as low as those typical for the ethanol-active class I mammalian enzyme and do not at all resemble those for class III, for which ethanol is hardly a substrate and pyrazole is hardly an inhibitor. Further out in the substrate pocket, several residues differ from the mammalian classes, affecting large substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequencing of a processed pseudogene derived from a human class III alcohol dehydrogenase gene

Current information on the molecular structure of human alcohol dehydrogenase (ADH) genes is fragmentary. To characterize all ADH genes, we have isolated 63 ADH clones from human genomic libraries made from one individual. Fifty-nine clones have been classified into five previously known loci: ADH1 (18 clones), ADH2 (20 clones), and ADH3 class I (16 clones), ADH4 class II (4 clones), and ADH5 class III (1 clone). Sequencing of one of the remaining four unclassified clones, SY lambda ADHE38, about 1.1 kb in length, shows no introns and three frameshift mutations in the coding region, with a total of 10 internal termination codons. When its deduced amino acid sequence was compared with those of the class I, class II, and class III ADHs, the proportions of identical amino acids were 56.7%, 55.5%, and 88.7%, respectively, suggesting that the processed pseudogene was derived from an ADH5 gene. The duplication event seems to have occurred about 3.5 million years ago, and the pseudogene has undergone a rapid change since then.     

Three-dimensional structures of the three human class I alcohol dehydrogenases

In contrast with other animal species, humans possess three distinct genes for class I alcohol dehydrogenase and show polymorphic variation in the ADH1B and ADH1C genes. The three class I alcohol dehydrogenase isoenzymes share approximately 93% sequence identity but differ in their substrate specificity and their developmental expression. We report here the first three-dimensional structures for the ADH1A and ADH1C*2 gene products at 2.5 and 2.0 A, respectively, and the structure of the ADH1B*1 gene product in a binary complex with cofactor at 2.2 A. Not surprisingly, the overall structure of each isoenzyme is highly similar to the others. However, the substitution of Gly for Arg at position 47 in the ADH1A isoenzyme promotes a greater extent of domain closure in the ADH1A isoenzyme, whereas substitution at position 271 may account for the lower turnover rate for the ADH1C*2 isoenzyme relative to its polymorphic variant, ADH1C*1. The substrate-binding pockets of each isoenzyme possess a unique topology that dictates each isoenzyme's distinct but overlapping substrate preferences. ADH1*B1 has the most restrictive substrate-binding site near the catalytic zinc atom, whereas both ADH1A and ADH1C*2 possess amino acid substitutions that correlate with their better efficiency for the oxidation of secondary alcohols. These structures describe the nature of their individual substrate-binding pockets and will improve our understanding of how the metabolism of beverage ethanol affects the normal metabolic processes performed by these isoenzymes.     

Histochemical assay to detect class III ADH activity in situ in Arabidopsis seedlings.

Whole-mount detection methods are quick, inexpensive and offer the possibility of studying the temporal and spatial patterns of gene expression in a morphological context. These methods have been used widely to detect messenger RNAs and to measure enzymatic activity of reporter genes, such as beta-galactosidase or beta-glucuronidase. Taking advantage of the fact that NADH generated during the oxidation of formaldehyde by class III alcohol dehydrogenase can reduce the compound nitroblue tetrazolium to form a blue precipitate, we have developed a new method to detect class III alcohol dehydrogenase activity in situ in whole Arabidopsis plants. This reaction has been used earlier for in situ electrophoresis detection and for histochemical analysis in animal tissue sections. With a few modifications, it can be used in whole Arabidopsis plants or excised plant tissues to allow a rapid analysis of class III ADH activity during development or in response to elicitors. The method might be extended to other dehydrogenases by using specific substrates.     

Characteristics of mammalian class III alcohol dehydrogenases, an enzyme less variable than the traditional liver enzyme of class I

Class III alcohol dehydrogenase, whose activity toward ethanol is negligible, has defined, specific properties and is not just a "variant" of the class I protein, the traditional liver enzyme. The primary structure of the horse class III protein has now been determined, and this allows the comparison of alcohol dehydrogenases from human, horse, and rat for both classes III and I, providing identical triads for both these enzyme types. Many consistent differences between the classes separate the two forms as distinct enzymes with characteristic properties. The mammalian class III enzymes are much less variable in structure than the corresponding typical liver enzymes of class I: there are 35 versus 84 positional differences in these identical three-species sets. The class III and class I subunits contain four versus two tryptophan residues, respectively. This makes the differences in absorbance at 280 nm a characteristic property. There are also 4-6 fewer positive charges in the class III enzymes accounting for their electrophoretic differences. The substrate binding site of class III differs from that of class I by replacements at positions that form the hydrophobic barrel typical for this site. In class III, two to four of these positions contain residues with polar or even charged side chains (positions 57 and 93 in all species, plus positions 116 in the horse and 140 in the human and the horse), while corresponding intraclass variation is small. All these structural features correlate with functional characteristics and suggest that the enzyme classes serve different roles. In addition, the replacements between these triad sets illustrate further general properties of the two mammalian alcohol dehydrogenase classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Maize glutathione-dependent formaldehyde dehydrogenase cDNA: a novel plant gene of detoxification

We have previously shown that intact plants and cultured plant cells can metabolize and detoxify formaldehyde through the action of a glutathione-dependent formaldehyde dehydrogenase (FDH), followed by C-1 metabolism of the initial metabolite (formic acid). The cloning and heterologous expression of a cDNA for the glutathione-dependent formaldehyde dehydrogenase from Zea mays L. is now described. The functional expression of the maize cDNA in Escherichia coli proved that the cloned enzyme catalyses the NAD⁺- and glutathione (GSH)-dependent oxidation of formaldehyde. The deduced amino acid sequence of 41 kDa was on average 65% identical with class III alcohol dehydrogenases from animals and less than 60% identical with conventional plant alcohol dehydrogenases (ADH) utilizing ethanol. Genomic analysis suggested the existence of a single gene for this cDNA. Phylogenetic analysis supports the convergent evolution of ethanol-consuming ADHs in animals and plants from formaldehyde-detoxifying ancestors. The high structural conservation of present-day glutathione-dependent FDH in microorganisms, plants and animals is consistent with a universal importance of these detoxifying enzymes.     

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.     

Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family

Summary Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in -crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II () and class III () ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (, , and ) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.Offprint requests to: B.V. Plapp     

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase.

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.     

Purification and comparative studies of alcohol dehydrogenases

Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies. The procedure achieves an 80-130 fold purification for animal enzymes. However, only a 5-15 fold purification for plant enzymes was attained because of the instability of these enzymes. Purified alcohol dehydrogenases from animal and plant sources differ in coenzyme and substrate specificities. The enzymes from mammalian, avian and fish livers display aldehyde oxidizing and esterolytic activities in addition to alcohol oxidizing activity. However, the enzymes from plants and yeast show only the oxidative activity toward alcohols. Chemical modifications have been performed to identify amino acid residues which are essential to the oxidative and esterolytic activities of alcohol dehydrogenases.     

Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells

One cell strain with stable tolerance to allyl alcohol (AA^r) was selected from 6 × 10⁸ suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA^r cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA^r cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells.     

Conservative evolution in duplicated genes of the primate Class I ADH cluster

Humans have seven alcohol dehydrogenase genes (ADH) falling into five classes. Three out of the seven genes (ADH1A, ADH1B and ADH1C) belonging to Class I are expressed primarily in liver and code the main enzymes catalyzing ethanol oxidization. The three genes are tandemly arrayed within the ADH cluster on chromosome 4 and have very high nucleotide similarity to each other (exons: >90%; introns: >70%), suggesting the genes have been generated by duplication event(s). One explanation for maintaining similarity of such clustered genes is homogenization via gene conversion(s). Alternatively, recency of the duplications or some other functional constraints might explain the high similarities among the genes. To test for gene conversion, we sequenced introns 2, 3, and 8 of all three Class I genes (total>15.0 kb) for five non-human primates--four great apes and one Old World Monkey (OWM)--and compared them with those of humans. The phylogenetic analysis shows each intron sequence clusters strongly within each gene, giving no evidence for gene conversion(s). Several lines of evidence indicate that the first split was between ADH1C and the gene that gave rise to ADH1A and ADH1B. We also analyzed cDNA sequences of the three genes that have been previously reported in mouse and Catarrhines (OWMs, chimpanzee, and humans) and found that the synonymous and non-synonymous substitution (dN/dS) ratios in all pairs are less than 1 representing purifying selection. This suggests that purifying selection is more important than gene conversion(s) in maintaining the overall sequence similarity among the Class I genes. We speculate that the highly conserved sequences on the three duplicated genes in primates have been achieved essentially by maintaining stability of the hetero-dimer formation that might have been related to dietary adaptation in primate evolution.     

Specificity of the dehydrogenases of maize endosperm

By means of starch gel electrophoresis and spectrophotometric assays, several different, specific dehydrogenases have been detected in liquid endosperm of maize 16 days after pollination. The typical alcohol dehydrogenase (ADH) bands on the starch gel appear when ethanol is used as substrate in the reaction mixture. However, some activity does appear with no substrate and with galactose or lactic acid as substrates, though not to the extent previously found and probably not due to the presence of a general type dehydrogenase as previously suggested (Scandalios, 1967). No specific activity appears with glucose, glucose 6-phosphate, galactose, malic acid, and isocitric acid when these are substituted for ethanol in the spectrophotometric assay for ADH. However, more specific spectrophotometric assays do show activity for malic acid and for isocitric acid. Both these enzymes, malate dehydrogenase (MDH) and isocitrate dehydrogenase, show definite patterns on the zymogram, with only a slight overlap with the ADH bands. MDH shows zymogram overlap with lactic acid as substrate.This work was supported by the U.S. Atomic Energy Commission, under contract No. AT(11-1)-1338.     

Class II alcohol dehydrogenase (ADH2)--adding the structure

Class II alcohol dehydrogenase (ADH2) represents a highly divergent class of alcohol dehydrogenases predominantly found in liver. Several species variants of ADH2 have been described, and the rodent enzymes form a functionally distinct subgroup with interesting catalytic properties. First, as compared with other ADHs, the catalytic efficiency is low for this subgroup. Second, the substrate repertoire is unique, e.g. rodent ADH2s are not saturated with ethanol as substrate, and while omega-hydroxy fatty acids are common substrates for the human ADH1-ADH4 isoenzymes, including ADH2, these compounds function as inhibitors rather than substrates. The recently determined structure of mouse ADH2 reveals a novel substrate-pocket topography that accounts for the observed substrate specificity and may, therefore, be important for the exploration of orphan substrates of ADH2. It is possible to improve the catalytic efficiency of mouse ADH2 by an array of mutations at position 47. Residue Pro47 of the wild type ADH2 enzyme seems to strain the binding of coenzyme, which prevents a close approach between the coenzyme and substrate for efficient hydrogen transfer. Based on crystallographic and mechanistic investigations, the effects of residue replacements at position 47 are multiple, affecting the distance for hydride transfer, the pK(a) of the bound alcohol substrate as well as the affinity for coenzyme.     

Hydrophobic anion activation of human liver chi chi alcohol dehydrogenase

Class III alcohol dehydrogenase (chi chi-ADH) from human liver binds both ethanol and acetaldehyde so poorly that their Km values cannot be determined, even at ethanol concentrations up to 3 M. However, long-chain carboxylates, e.g., pentanoate, octanoate, deoxycholate, and other anions, substantially enhance the binding of ethanol and other substrates and hence the activity of class III ADH up to 30-fold. Thus, in the presence of 1 mM octanoate, ethanol displays Michaelis-Menten kinetics. The degree of activation depends on the size both of the substrate and of the activator; generally, longer, negatively charged activators result in greater activation. At pH 10, the activator binds to the E-NAD+ form of the enzyme to potentiate substrate binding. Pentanoate activates methylcrotyl alcohol oxidation and methylcrotyl aldehyde reduction 14- and 30-fold, respectively. Such enhancements of both oxidation and reduction are specific for class III ADH; neither class I nor class II shows this effect. The implications as to the nature of the physiological substrate(s) of class III ADH are discussed in light of the recent finding that this ADH and glutathione-dependent formaldehyde dehydrogenase are identical. A new rapid purification procedure for chi chi-ADH is presented.     

Cloning and analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cerevisiae.

A gene (FDH1) of Candida maltosa which confers resistance to formaldehyde in Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The gene has a single intron which possesses the highly conserved splicing signals found in S. cerevisiae introns. We demonstrated that processing of the pre-mRNA of the cloned gene occurred identically in both S. cerevisiae and C. maltosa. The predicted amino acid sequence from the cloned gene showed 65.5% identity to human alcohol dehydrogenase (ADH) class III and 23.9% identity to S. cerevisiae ADH1. The most probable mechanism of resistance to formaldehyde is thought to be the glutathione-dependent oxidation of formaldehyde which is characteristic for ADH class III. The cloned FDH1 gene was successfully employed as a dominant selectable marker in the transformation of S. cerevisiae.     

Alcohol dehydrogenase in the mouse epididymis. Genetic variation and cellular localization

The cellular localization of alcohol dehydrogenase (ADH) in the mouse epididymis was investigated using differential substrate specificities and genetic variation as a means of distinguishing these enzymes histochemically in tissue sections. ADH-C2 exhibited high activity in BALB/c epididymis and was observed as a discrete zone within duct epithelial cells near the nuclei. This isozyme exhibited no detectable activity in C57BL/6J epididymis extracts or histochemical sections.