STRUCTURE DETERMINATION OF CHOLESTEROL OXIDASE CONTAINING COVALENTLY BOUND FAD. Kimberley Q. Yue, Nathalie Croteau and Alice Vrielink, Biochemistry Department & Montreal Joint Centre for Structural Biology, McGill University, Montreal, Quebec, Canada.

Cholesterol oxidase ia a bifunctional flavoenzyme which catalyses the oxidation of steroids containing a ß-hydroxyl group and the isomerization of the double bond at [[Delta]]⁵-[[Delta]]⁶ of the steroid ring. The protein is used clinically in the determination of serum cholesterol and for the assessment of arteriosclerosis. The structure of a form of the FAD prosthetic group non covalently bound to the enzyme has been solved and refined both in the presence and absence of a bound steroid substrate (1,2). A second form of the enzyme has been obtained from Brevibacterium sterolicum containing FAD covalently linked to His121 via the C8[[alpha]] group of the flavin isoalloxazine ring. Structural analyses of both forms of the enzyme will provide a unique opportunity to study the relationships between the flavin environment and their redox potential.

Single rod shaped crystals have been obtained for both the native enzyme containing covalently bound FAD and the His121Ala mutant. These crystals are grown by vapour diffusion using the hanging drop technique. The precipitant conditions are 12% PEG8K, 75mM MnSO₄, 100mM cacodylate pH 5.2. In order to obtain large single crystals, a temperature difference between the nucleation event and the growth event is essential. The space group of cholesterol oxidase is monoclinic P2₁. The cell dimensions are a=77.6Å, b=125.7Å, c=81.5Å and ß=109.1deg. with two molecules per asymmetric unit. X-ray data collection is carried out by flash-cooling the crystals to 115K in a nitrogen stream. One heavy atom derivative has been obtained by soaking the crystals in 0.5mM K₂Pt(CN)₄. Further screening for heavy atom derivatives is in progress.

References:

1. Vrielink, A. Et al., (1991). J.Mol.Biol. 219, 533-544.

2. Li, J. Et al., (1993). Biochemistry 32, 11507-11515.