Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase.
Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, 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 appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation 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 structure of the enzyme constructed on the basis of homology modeling.
Yamashita
M
M
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Toyama
M
M
Ono
H
H
Fujii
I
I
Hirayama
N
N
Murooka
Y
Y
D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002789:Cholesterol Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D007536:Isomerism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D014961:X-Ray Diffraction
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D002789:Cholesterol Oxidase