Crystal structure of cholesterol oxidase complexed with a steroid substrate: Implications for flavin adenine dinucleotide dependent alcohol oxidases

• Published in: Biochemistry (Easton) 1993-11, Vol.32 (43), p.11507-11515
• Main Authors: Li, Jiayao, Vrielink, Alice, Brick, Peter, Blow, David M.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Brevibacterium - enzymology; Cholesterol Oxidase - chemistry; Cholesterol Oxidase - metabolism; Crystallography, X-Ray; Dehydroepiandrosterone - metabolism; Enzymes and enzyme inhibitors; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Models, Molecular; Molecular Conformation; Oxidoreductases; Protein Binding; Protein Conformation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00094a006

Cholesterol oxidase from Brevibacterium sterolicum is a flavin-dependent enzyme that catalyzes the oxidation and isomerization of 3 beta-hydroxy steroids with a double bond at delta 5-delta 6 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-A resolution. The final crystallographic R-value is 15.7% for all reflections between 10.0- and 1.8-A 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.