CRYSTAL-STRUCTURE OF CHOLESTEROL OXIDASE COMPLEXED WITH A STEROID SUBSTRATE - IMPLICATIONS FOR FLAVIN ADENINE-DINUCLEOTIDE DEPENDENT ALCOHOL OXIDASES

Cholesterol oxidase from Brevibacterium sterolicum is a flavin-dependent enzyme that catalyzes the oxidation and isomerization of 3beta-hydroxy steroids with a double bond at DELTA5-DELTA6 of the steroid ring backbone. The crystal structure of the free enzyme in the absence of a steroid substrate has previously been determined. In this paper we report the crystal structure of the complex of cholesterol oxidase with the steroid substrate dehydroisoandrosterone, refined at 1.8-angstrom resolution. The final crystallographic R-value is 15.7% for all reflections between 10.0- and 1.8-angstrom resolution. The steroid is buried within the protein in an internal cavity which, in the free enzyme crystal structure, was occupied by a lattice of water molecules. The conformations of a number of side chains lining the active-site cavity have changed in order to accommodate the steroid substrate. A loop region of the structure between residues 70 and 90 differs significantly between the substrate-free and substrate-bound forms of the enzyme, presumably to facilitate binding of the steroid. The hydroxyl group of the steroid substrate is hydrogen-bonded to both the flavin ring system of the FAD cofactor and a bound water molecule. FAD-dependent cholesterol oxidase shares significant structural homology with another flavoenzyme, glucose oxidase, suggesting that it might also be a member of the glucose-methanol-choline (GMC) oxidoreductase family. Although there is only limited sequence homology, a superposition of these two structures reveals a conserved histidine residue within hydrogen-bonding distance of the active-site water molecule. This histidine residue is completely conserved in the known sequences of GMC oxidoreductases and may act as the general base in the oxidation reaction. The structure of the substrate complex suggests possible mechanisms for the oxidation reaction, although the isomerization reaction cannot be explained by an analysis of the complex.