Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-Å resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu 475 , located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A waterfilled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.Cholesterol oxidase is a flavoprotein that catalyzes the oxidation and isomerization of steroids containing a 3␤-hydroxyl group and a double bond at C-5 of the steroid ring system (see Scheme 1). The enzyme has been used in the determination of serum cholesterol and in the clinical diagnosis of arteriosclerosis and other lipid disorders. In addition, it has been shown to be a potent larvicide (1-3) and is currently being developed in the agricultural industry as a pest control (4). Furthermore, cholesterol oxidase is an example of a soluble enzyme that interacts with a lipid bilayer to bind an insoluble substrate. Structural and biochemical studies on the enzyme containing the flavin adenine dinucleotide (FAD) 1 cofactor non-covalently bound to the protein have revealed the region of the enzyme involved in interaction with the lipid bilayer and have led to a possible mechanism for membrane interaction (5-7).In Brevibacterium sterolicum, cholesterol oxidase has been found to exist in two forms, one in which the FAD cofactor is non-covalently bound to the enzyme (BCO1) and one in which the cofactor is covalently linked (BCO2). Although these enzymes share the same catalytic activity they show no sequence homology. Comparisons of these two forms of cholesterol oxidase reveal large differences in redox potential and kinetic properties suggesting different mechanisms (8). These studies support the hypothesis that there are significant structural differences between the two enzyme forms, correlated with changes in biochemical properties.Here we report the structure of BCO2 refined to 1.7-Å resolution. The structure reveals featur...

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