Mutational analysis of the oxygen-binding site of cholesterol oxidase and its impact on dye-mediated dehydrogenase activity

Abstract

Cholesterol oxidases (ChOx, EC 1.1.3.6) is the name of the group of flavin adenine dinucleotide (FAD)-containing enzymes, catalyzing the oxidation of cholesterol using oxygen as the electron acceptor. ChOx is widely used for the determination of cholesterol concentration in the serum and in other clinical samples. The development of an enzyme-based sensor employing the enzyme ChOx has great potential as a simple and economical sensor system. However, an electron mediator-type electrochemical monitoring system based on oxidases is inherently affected by dissolved oxygen. Therefore, oxidases that are less O2-sensitive would be very advantageous for the development of amperometric enzyme sensors using artificial electron acceptors. In this study, we carried out site-directed mutagenesis on oxygen-binding residues, which are observed in the high-resolution crystal structure, in order to elucidate the amino acid residues responsible for the oxidase activity. First, Ala substitutions were carried out. Among the 6 Ala-substituted ChOxs, along with saturation mutagenesis, Val191Ala showed a significant decrease in its oxidase activity, but showed increased dehydrogenase activity. The dehydrogenase/oxidase ratio of Val191Ala was more than 150%, which was a 400-fold increase compared with the ratio for the wild-type enzyme. This engineering strategy will provide an ideal enzyme for electrochemical monitoring of cholesterol.