Mammalian class IV alcohol dehydrogenase (stomachalcohol dehydrogenase): structure, origin, and correlation withenzymology.

Abstract

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; -68% residue identities) than to the other classes (II, m, and V; -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 kalt 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.

Molecular characterization of a class IV human alcohol dehydrogenase gene (ADH7)

“…Vertebrate ADHs considered were class I ADH~ (HsaA), fl (HsaB), ~ (HsaC) from human and those from baboon (PhaB), rhesus monkey (MacA), mouse (MmuA), rat (RnoA), quail (Cja), chicken (Gga), and frog (Rpe), and ADH-E (EcaE) and ADH-S (EcaS) from horse, one class II ADH from human (HsaP), class III ADHs from human (HsaX), mouse (MmuX), rat (RnoX) and horse (EcaX), and class V ADH (Hsa6) deduced from human ADH6 ( [6] and references therein). Furthermore, ADH1 (PmaA) and ADH2 (PmalV) from deer mouse (Peromyscus maniculatus) [14] and class IV ADH from rat (RnolV) [8] were also included.…”

“…By comparing RnolV to other classes of ADHs, amino acid residues Gly (47), Ala (294), and Tyr (363) of RnolV are suggested to be class IV-specific changes by affecting both substrate and coenzyme binding [8]. However, the corresponding amino acids for Hsa7 are Arg (48), Val (294), and Asn (363) and the three residues are not shared by the two class IV ADHs.…”

“…The class V protein has been deduced from genomic and cDNA clones [5], but the protein has not been isolated from natural sources. The class IV protein appears to be expressed mainly in the stomach mucosa [4,8]. The molecular structure of a class IV protein from rat has been characterized only recently [8], but that for human is not known.…”

“…The class IV protein appears to be expressed mainly in the stomach mucosa [4,8]. The molecular structure of a class IV protein from rat has been characterized only recently [8], but that for human is not known. We report here the cloning and sequencing of a novel human ADH gene (ADH7).…”

Diversity of Vertebrate Class I Alcohol Dehydrogenase

“…At least four further classes of alcohol dehydrogenase exist, indicating still more gene duplications, giving rise to classes I1 [2], IV [S], V [9] and VI [lo], of which just a few variants are known, not allowing for extensive conclusions on their patterns of divergence. However, emerging data on class IV suggest this stomach enzyme to have a duplicatory origin from class I [8] and a variability in between that of classes I and 111 [ll]. Nomenclature of the alcohol dehydrogenases, in particular of the recently established classes IV, V and VI, is still not unanimously accepted, but has been recently summarized [12], with the numbering system now used being the one then suggested.…”