Lysine mutagenesis identifies cationic charges of human CYP17 that interact with cytochrome b5 to promote male sex-hormone biosynthesis

Human CYP17 (17α-hydroxylase-17,20-lyase; also cytochrome P450c17 or cytochrome P450_17α) catalyses a hydroxylation reaction and another reaction involving the cleavage of a C-C bond (the lyase activity) that is required only for androgen production. Single amino acid mutations in human CYP17, Arg³⁴⁷ → His and Arg³⁵⁸ → Gln, have been reported to result in the loss of the lyase activity and to cause sexual phenotypic changes in 46XY male patients. By using site-directed mutagenesis we show here that another mutation in human CYP17, Arg⁴⁴⁹ → Ala, for which human variants have yet not been described, also leads to selective lyase deficiency. Furthermore, all the three types of mutants display a loss of responsiveness to cytochrome b₅, an interaction that is essential for lyase activity, and hence male sex-hormone biosynthesis. That the defect could be essentially reversed by lysine mutagenesis has led to the conclusion that the cationic charges on all three residues (at the positions of Arg³⁴⁷, Arg³⁵⁸, Arg⁴⁴⁹) are vital for the functional interaction of CYP17 with cytochrome b₅ and that the loss of any one of these cationic charges is catastrophic.