Alcohol Dehydrogenase Class III Contrasted to Class I

Abstract

Alcohol dehydrogenases of classes I (the classical liver enzyme) and I11 (formaldehyde dehydrogenase) constitute a pair of moderately related enzymes (63% residue identity between the human forms) that differ fundamentally in many respects. To elucidate the nature of the differences, we have characterized alcohol dehydrogenase from the most primitive vertebrate line (a cyclostome, Atlantic Hagfish), related that to the multiplicity of the human enzyme, and submitted the enzymes to in vitro hybridization for evaluation of subunit interactions. Three findings illustrate important principles of the enzyme system. First, the alcohol dehydrogenase purified from cyclostomes is a class-I11 protein, compatible with the facts that cyclostomes constitute the earliest extant vertebrate line and that class I11 has a distant pre-vertebrate origin. Second, the hagfish enzyme shows multiplicity, with acidic forms in decreasing yield and with amino acid sequences identical between two major isoforms, both aspects constituting properties similar to those of the corresponding human forms. The chemically different subunits are present as homodimers and heterodimers of unmodified and modified subunits, suggesting that the class-I11 multiplicity derives from modification of a type common to lines as divergent as mammals and cyclostomes. Third, the human enzyme can form cross-species hybrid dimers in vitro with the cod and hagfish or Drosophilu class-I11 enzymes (positional identity with the human form of 82, 76 and 70%, respectively). Hence, the results provide experimental evidence for little class-I11 divergence in the segments of subunit interactions. The extent of conservation of residues directly involved in the formation of the subunit interface also reveals a clearly different pattern between classes I and 111. This highlights separation of divergent forms in an enzyme system, with the constant form (class 111) resembling house-keeping enzymes, and exhibiting a correlation between subunit-interacting and substrate-interacting segments.Mammalian alcohol dehydrogenases of apparently six different classes are known [l]. They differ considerably in primary structure (sequence identity, 50-67 %), substrate specificity, tissue distribution and genetic regulation. Knowledge is by far most extensive for the class-I and class-I11 enzymes. The former is the classical liver alcohol dehydrogenase, with considerable ethanol dehydrogenase activity, while the latter constitutes the glutathione-dependent formaldehyde dehydrogenase. Multiple forms within both classes increase the complexity of the enzyme system still further.The class-I enzyme exhibits multiplicity at different levels. One level is derived from gene duplications [2], with resultant subunits explaining the isozymes, long known, for the human enzyme [3]. Another level derives from allelic variability [4], while a third is explained by secondary modifications that have been characterized to only a limited extent in humans and some mammals [3,. The mammalian class-I11 enzymes als...

Crystal structure of cod liver class I alcohol dehydrogenase: Substrate pocket and structurally variable segments

“…They are the classical liver alcohol dehydrogenase (class I type) and the ubiquitous, glutathione-dependent formaldehyde dehydrogenase (class 111 type). The two classes are derived from a gene dupli- cation, and the original class is thought to be like the class I11 enzyme, because animals of early origin, such as cyclostomes and invertebrates, have this form, but appear to lack the class I form (Kaiser et al, 1993;Danielsson et al, 1994aDanielsson et al, . 1994b.…”

Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis

“…Reapplication of the filter to the sequencer gave the result Ser-Thr-Ala-Gly-Lys, which accounts for the amino acid composition of the peptide. Similarly, the N-terminal sequence of hagfish alcohol dehydrogenase was interpreted for 14 residues (Table 2) at 3% initial yield of an N-terminal peptide generated by GluC digestion of the intact protein [11]. Before that, the sample was analyzed by five cycles of Edman degradation which established its blocked nature, after which the filter (Polybrene-treated glass fiber) was removed and the polypeptide in situ deacetylated followed by reapplication to the sequencer.…”

“…The polypeptides employed were obtained from natural sources [6][7][8][9][10][11] or prepared synthetically [15]. Samples for deacetylation were either recovered in solution from column separations and lyophilized in glass tubes, directly spotted onto sequencer filters, or electroblotted to sequencer filters after SDS-polyacrylamide gel separation [16].…”

Alcohol dehydrogenase of class III: consistent patterns of structural and functional conservation in relation to class I and other proteins

“…Structurally, variability patterns are consistent throughout the vertebrate system with a ratio in accepted point mutations versus class I of 0.4. This ratio between different classes of a zinc enzyme is comparable to that between different heme proteins (cytochrome c and myoglobin), suggesting defined but non-identical functions also for the alcohol dehydrogenase classes.Key words: Alcohol dehydrogenase; Reptilian protein; Uromastix hardwickii; Amino acid sequence; Molecular evolution absence of class I in species diverging before the supposed class III duplication [6,9], and presence of enzymes with class-mixed properties in species originating at that time (fish [10]).Although the alcohol dehydrogenase classes now constitute a model system with an apparent class III parent form and with repeated descendents from separate gene duplications, class III has not been characterized in any sub-mammalian traditional vertebrate (Superclass Gnathostomata). Many of the conclusions therefore depend on the recently characterized insect enzyme [6] and cannot be directly correlated with variants of class I which have been studied in sub-mammalian vertebrate lines.…”

“…Key words: Alcohol dehydrogenase; Reptilian protein; Uromastix hardwickii; Amino acid sequence; Molecular evolution absence of class I in species diverging before the supposed class III duplication [6,9], and presence of enzymes with class-mixed properties in species originating at that time (fish [10]).…”