Liver Class‐I Alcohol Dehydrogenase Isozyme Relationships and Constant Patterns in a Variable Basic Structure

Abstract

The major ethanol dehydrogenase of cobra liver was characterized in order to clarify isozyme relationships and functional motifs of the vertebrate enzyme. The cobra protein is a class-I form, most related to one of the isozyme subunits (the a form) in Uromastix (lizard) liver. This positions the isozyme duplication and defines the main-line alternative. The new structure also allows extensive correlations with structure/function relationships for alcohol dehydrogenases in general, of which 38 animal variants (still disregarding strain and allelic differences) now have been characterized. Architectural features are discerned, distinguishing the enzyme at large, the classes, and the functional interactions at the sites of substrate binding and coenzyme binding. Variability is greater at the substrate-binding site, with only one of 13 residues strictly conserved (His67, one of the active-site zinc ligands) but all other residues differing among and frequently within classes. However, many substrate-interacting residues are class preferential and may be used in predictive assignments. Class-I/III differences concern position 48 (typically Ser in (residues 178, 203, 223, 224, 228; capital letters for residues strictly conserved and small-cases letters for residues nearly so). This motif is class independent and unique to animal alcohol dehydrogenases. Therefore, the novel enzyme structure establishes class-I isozyme relationships, shows characteristic 'constant' residues also in the 'variable' class-I line, and defines residue-specific patterns which may have a predictive value in functional assignments of an increasing number of undefined further forms expected to result from gene projects.

Arabidopsis Formaldehyde Dehydrogenase

“…The ethanolactive class P enzymes from Arubidopsis and pea are significantly more related to their respective class 111 forms (58-59%) than to the class I forms (47-51 %). Taken together these results confirm that class 111 constitutes a divergent family, common to plants and animals, while the ethanol-active forms from plants (class P) and animals (class I) constitute separate structures (Shafqat et al, 1996a). Similar conclusions are obtained by a close evaluation of the residues at the active site (Fig.…”

“…Thus, they appear to form separate lines within the family of MDR (medium-chain dehydrogenased reductases) enzymes and exhibit functional convergence toward ethanol dehydrogenase activity. This result also positions the class 111 form as an apparent ancestor in common (Danielsson and Jornvall, 1992;Shafqat et al, 1996a), compatible with the ubiquitous presence of that activity Danielsson et al, 1994). Overall, the alcohol dehydrogenase constitutes a complex enzyme system, where related enzymes Correspondence to X.…”

“…Ethanol-active alcohol dehydrogenases in plants, although related to those in animals (BriindCn et al, 1984;Chang and Meyerowitz, 1986;Yokoyama and Harry, 1993), were recently shown to originate through separate gene duplications from the formaldehyde dehydrogenase (class 111 alcohol dehydrogenase) line (Shafqat et al, 1996a). Thus, they appear to form separate lines within the family of MDR (medium-chain dehydrogenased reductases) enzymes and exhibit functional convergence toward ethanol dehydrogenase activity.…”

“…[the ethanol dehydrogenase of liver (class I) and plants (class P)] with similar conformations and activities exhibit molecular differences and appear to be paralogous, whereas the formaldehyde dehydrogenases (class I11 alcohol dehydrogenases) from the same sources form a third pattern and origin, in apparent orthologous relationships (Shafqat et al, 1996a).…”

“…Regarding classes I and P, many species variants have long been known (Sun and Plapp, 1992;Yokoyama and Harry, 1993;Persson et al, 1993;Shafqat et al, 1996b), although the internal patterns of class I/P molecular variation have not been compared. Regarding class I11 in plants, glutathione-dependent formaldehyde dehydrogenase activity has been reported for a few species (Uotila and Koivusalo, 1979;Giese et al, 1994) and recently also amino acid sequences for pea and maize class 111 alcohol dehydrogenases (Shafqat et al, 1996a;Sugaya and Sakai, 1996), but further variants and intra-class divergence are unknown. We have now characterized Arubidopsis class I11 alcohol dehydrogenase at both the protein and cDNA levels.…”