Separation of the two reactions, oxidation and isomerization, catalyzed byStreptomyces cholesterol oxidase

Site-directed mutagenesis was used to identify key amino acid residues of t he cholesterol oxidase from Streptomyces sp,, which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one, Eight mutant en zymes were constructed and the following amino acid substitutions were iden tified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q, Mutants N318A and N318H retained both oxidation and isomerization activities. The m utant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained th eir isomerization activities. The two catalytic reactions, oxidation and is omerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appe ar to participate in the catalysis of cholesterol oxidase, whereas N318 doe s not. An analysis of the products of these mutant enzymes suggested that t he previously proposed 6-hydroxylation reaction by cholesterol oxidase is a ctually autooxidation from 5-cholesten-3-one, Kinetic studies of the purifi ed wild-type and mutant enzymes showed that the k(cat)/K-m values for oxida tion in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional struct ure of the enzyme constructed on the basis of homology modeling.