The amino acid residues affecting the activity and azole susceptibility ofrat CYP51 (Sterol 14-demethylase p450)

The amino acid residues affecting the function of rat sterol 18-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Esche richia coli, Substitution of highly conserved Y131, E369, R372, or R382 dec reased the expression of CYP51 protein, indicating some structural importan ce of these residues. Substitution of H314, T315, or S316 caused considerab le effects on the catalytic activity, and T315 was identified as the "conse rved threonine" of CYP51. H314 was important for maintenance of the activit y of CYP51 and was a characteristic residue of this P450, because the posit ion corresponding to this residue is occupied by an acidic amino acid in mo st other P450 species. A144 was identified as a residue affecting the inter action of CYP51 with ketoconazole. Substitution of A144 with I, which occup ies the corresponding position in fungal CYP51, enhanced the ketoconazole s usceptibility of rat CYP51 with little change in the catalytic activity, in dicating an important role of this residue in determination of the ketocona zole susceptibility of CYP51. Alteration of the catalytic activity was caus ed by the substitution at some other sites, whereas substitution of a few h ighly conserved amino acids caused little alteration of the activity of CYP 51.