Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Abstract

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His 69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (ϳ3 versus ϳ5 M, respectively), and the mutant protein had a melting temperature ϳ10 -15°C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 M urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.Bacterial cholesterol oxidase (CO, EC 1.1.3.6) is a monomeric bifunctional FAD-containing flavoenzyme that catalyzes the oxidation of 3␤-hydroxysteroids and the isomerization of the intermediate, ⌬ 5-6 -ene-3␤-ketosteroid (cholest-5-en-3-one) to produce ⌬ 3-4 -ene-3␤-ketosteroid (cholest-4-en-3-one). Bacteria that produce CO can be classified into two classes: non-pathogenic (e.g. Streptomyces and fast-growing Mycobacteria; these bacteria utilize cholesterol as their carbon source) and pathogenic bacteria (e.g. Rhodococcus equi and slow-growing Mycobacteria). Pathogenic bacteria require CO for infection of the host macrophage probably because of its ability to alter the physical structure of the membrane by converting cholesterol into cholest-4-en-3-one.The crystal structure of COs belonging to two distinct structural classes (1) have been solved by the Vrielink laboratory (2-4). Members classified as type I, such as those from Streptomyces sp. SA-COO and R. equi (5) have their cofactor tightly but non-covalently bound to the enzyme. A second CO from Brevibacterium sterolicum classified as type II, is completely different. In this enzyme the cofactor is covalently linked to the protein via a bond between the 8-methyl group of the isoalloxazine ring and the ND1 atom of His 69 .Although COs of class I an...

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