Abstract
Cholesterol oxidase is a member of the glucose-methanol-choline (GMC) oxidoreductase family that is characterized by a conserved topology. We review our investigations into the reactivity of the Streptomyces cholesterol oxidase cofactor, flavin adenine dinucleotide (FAD), and the role of active-site residues. All of our mutagenesis, enzyme inhibition, and kinetic data demonstrate that the cofactor catalyzes oxidation of alcohols to ketones, but not oxygenation of carbon. Cholesterol oxidase catalyzes two reactions, oxidation and isomerization, in one active site, presumably because of the susceptibility of the reaction intermediate cholest-5-en-3-one to radical oxidation. This bifunctionality is not a shared characteristic with other GMC oxidoreductase family members. Furthermore, we have characterized the unusual inactivation of FAD by electrophilic substitution at C6 of the isoalloxazine ring upon ring opening of a cyclopropyl steroid. Another member of the GMC oxidoreductase family, methanol oxidase, is also inactivated by a cyclopropanol suggesting that inhibition by cyclopropanol inhibitors may be diagnostic of membership in this family.