Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidatio n and isomerization of cholesterol to cholest-4-en-3-one. The enzyme intera cts with lipid bilayers in order to bind its steroid substrate. The X-ray s tructure of the enzyme from Brevibacterium sterolicum revealed two loops, c omprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate [Vrielink et al. (1991) J. Mel. Biol. 219, 533-554; Li et al. (1993) Biochemistry 32, 11507-11515] . It was postulated that these loops must open, forming a hydrophobic chann el between the membrane and the active site of the protein and thus sequest ering the cholesterol substrate from the aqueous environment. Here we descr ibe the three-dimensional structure of the homologous enzyme from Streptomy ces refined to 1.5 Angstrom resolution. Structural comparisons to the enzym e from B, sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-8 7 adopts a small amphipathic helical turn with hydrophobic residues directe d toward the active site cavity and hydrophilic residues directed toward th e external surface of the molecule. It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate. Cons equently, the Streptomyces enzyme is a more efficient catalyst. In addition , we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His 447Gln) or the isomerization (Glu361Gln). Our structural and kinetic data i ndicate that His447 and Glu361 act as general base catalysts in association with conserved water H(2)O541 and Asn485. The His447, Glu361, H(2)O541 and Asn485 hydrogen bond network is conserved among other oxidoreductases. Thi s catalytic tetrad appears to-be a structural motif that occurs in flavoenz ymes that catalyze the oxidation of unactivated alcohols.