title: High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization.
abstract: Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features.
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia.
authors.forename: Emily
authors.initials: E
authors.lastname: Golden
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia.
authors.forename: Amir
authors.initials: A
authors.lastname: Karton
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia.
authors.forename: Alice
authors.initials: A
authors.lastname: Vrielink
mesh_terms: D002789:Cholesterol Oxidase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology*
chemical_list: D002789:Cholesterol Oxidase
keywords: cholesterol oxidase; redox stabilization