Library screening studies to investigate substrate specificity in the reaction catalyzed by cholesterol oxidase

We tested whether it is possible to alter the substrate specificity of cholesterol oxidase for similarly sized sterols, i.e. cholesterol, β-sitosterol and stigmasterol. Using existing X-ray crystal structures, we made a model of the predicted Michaelis complex of cholesterol and cholesterol oxidase. Based on this model, we identified five residues that are in direct contact with the steroid tail, Met58, Leu82, Val85, Met365 and Phe433. We prepared seven mutant libraries that contained the codon NYS (N = A, C, G, T; Y = C, Y; S = C, G) at one, two or three of the targeted positions by cassette mutagenesis. The libraries were screened for catalytic activity against three different sterols under k_cat*/K_m* conditions with 25 mol% sterol/DOPC unilamellar vesicles. The results of our screens suggest that specific packing interactions are not realized in the transition state of binding and that loss of active site water may be the predominant source of binding energy.