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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
J
J
Blackett Laboratory, Imperial College, London, England.
Vrielink
A
A
Brick
P
P
Blow
D M
DM
D001951:Brevibacterium / Q000201:enzymology; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D003687:Dehydroepiandrosterone / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation
D005182:Flavin-Adenine Dinucleotide; D003687:Dehydroepiandrosterone; D002789:Cholesterol Oxidase