Cholesterol oxidase from Brevibacterium sterolicum and Streptomyces hygroscopicus : a covalent FAD binding vs. a non-covalent one

Introduction Cholesterol oxidase (CO, EC 1.1.3.6) is a flavoenzyme which catalyzes the first step in the pathway of cholesterol degradation in various microorganisms. CO is a bifunctional enzyme: it catalyzes the oxidation of ß-hydroxysteroids and the isomerization of the produced [\s-ketosteroid to the [\4-3-ketosteroid. The three dimensional structure of CO from Brevibacterium, which contains non-covalently linked FAD, has been reported (1). Intriguingly, a second CO from a different strain of Brevibacterium contains a covalently linked 8-histidyl-FAD. We compare here two related COs, one from S. hygroscopicus (SCO) containing a freely dissociable cofactor, the other a recombinant protein from B. sterolicum (BCO) which contains covalently linked FAD and whose three-dimensional structure is elose to completion (A.Vrielink, personal communication). The redox and general properties of these two enzymes have been described recently. Marked differences between the two COs are the midpoint potentials and their reactivity towards sulfite (2). Further, we have studied a mutant form of BCO (H69A) obtained by site-directed mutagenesis, in which 8a-NI-histidyl-FAD bond cannot form. Although substantial advances have been made in recent years in understanding the mechanism of flavin attachment to protein, the role of the covalent link in the biological function is obscure (for a review see (3)). The aims of the present study is to assess the role of the FAD covalent link in CO by investigation of the stability and of some kinetic properties of these COs.