Medium-chain alcohol dehydrogenases : Studies of novel class I, II, III and IV forms revealing structural, functional, and evolutionary properties of this enzyme system

Sammanfattning: Novel forms of alcohol dehydrogenases of classes I, II, III, and IV were characterized. Together with other variants, they give useful information on molecular properties and functions of the alcohol dehydrogenase system. They further illustrate general aspects of protein variability, isozyme formation, and evolution of enzyme functions. Liver alcohol dehydrogenase of class I exhibits similar patterns of function and evolutionary speed in different lines (human/mammalian versus non-mammalian). This suggests that the enzyme is of equal importance in both lines supporting a common role. Similar patterns of variability are obtained from analysis of a structural RNA, linking protein and RNA data in a consistent manner. Comparisons of the variability between classes I and III show that class III evolves at a speed of about 0.4 that of class I, largely independent of lines compared. The pattern is highly similar to that of other well-studied proteins, such as myoglobin and cytochrome c, further strengthening the conclusion of a common role of class I. Determination of the N-terminal segments of alcohol dehydrogenase shows that the enzyme generally is acetylated in eukaryotes, and that the classes differ in a consistent manner, which is in marked contrast to their overall properties. Thus, the "variable" class I enzyme has the most conserved N-terminal segment. In contrast, that segment is divergent in the "constant" class III enzyme, suggesting an extra importance of the segment in class I. Isozyme variability was detected in a sub-mammalian line, and concluded to be the result of yet another gene duplication. The two isozyme subunits exhibit 19% differences, much more than between the mammalian isozymes, and this is in fact more than half of the variability between classes. Although both subunits are equally related to the human class I enzymes, analysis of the enzyme from a related line shows which of the two isozyme subunits that represents the main-line. The isozyme variation along the protein chain shows a pattern which has a general resemblance with the class I and inter-class variability, rather than with the pattern of the "constant" class III. Thus, ~30% of all isozyme differences coincide with the inter-class variation at functional segments. It can be concluded that the basic process of class evolution is similar in nature to that of isozyme evolution. Isozymes, classes and novel enzymes are related in a continuous flow of structural divergence and emergence of novel functions. A non-mammalian alcohol dehydrogenase of class II was found to have mixed-class properties, being functionally of class I type but structurally of class II type, with which it clusters in phylogenetic trees of vertebrate alcohol dehydrogenases, reflecting the separate duplicatory origin of class II and emergence of novel enzyme activities. The class II enzyme is found to be even more variable than class I. Furthermore, its intra-molecular patterns of variation resemble those of class I with segments of maximal variation at functionally important parts. The gastric alcohol dehydrogenase with its distinct enzymatic properties was concluded to constitute a separate class, class IV, which has emerged from the class I line through a separate gene duplication. The high kcat for ethanol is correlated with replacements for uncharged residues at positions 47 and 363, of importance for binding of the coenzyme. The high Km of the enzyme for ethanol is correlated with a small residue in the middle segment of the substrate-binding cleft. Similarly, a small residue in the inner segment of the substrate-binding cleft in one of the class I enzymes is also correlated with a relatively high Km for ethanol. In summary, the natural alcohol dehydrogenase variants have traced novel gene duplications, linked the isozyme emergence to that of classes, and correlated molecular variability patterns with protein function. Keywords: alcohol dehydrogenase, isozymes, classes, enzymes, amino acid sequence, enzyme functions, protein variability, evolution Lars Hjelmqvist: Medium-chain alcohol dehydrogenases. Studies of novel class I, II, III and IV forms revealing structural, functional, and evolutionary properties of this enzyme system; Stockholm 1995; ISBN 91-628- 1802-3