Lysine mutagenesis identifies cationic charges of human CYP17 that interact with cytochrome b(5) to promote male sex-hormone biosynthesis

Human CYP17 (17 alpha-hydroxylase-17,20-lyase; also cytochrome P450c17 or c ytochrome P450(17 alpha)) catalyses a hydroxylation reaction and another re action involving the cleavage of a C-C bond (the lyase activity) that is re quired only for androgen production. Single amino acid mutations in human C YP17, Arg(347)--> His and Arg(358) --> Gln, have been reported to result in the loss of the lyase activity and to cause sexual phenotypic changes in 4 6XY male patients. By using site-directed mutagenesis we show here that ano ther mutation in human CYP17, Arg(449) --> Ala, for which human variants ha ve yet not been described, also leads to selective lyase deficiency. Furthe rmore, all the three types of mutants display a loss of responsiveness to c ytochrome b(5), an interaction that is essential for lyase activity, and he nce male sex-hormone biosynthesis. That the defect could be essentially rev ersed by lysine mutagenesis has led to the conclusion that the cationic cha rges on all three residues (at the positions of Arg(347), Arg(358), Arg(449 )) are vital for the functional interaction of CYP17 with cytochrome b(5) a nd that the loss of any one of these cationic charges is catastrophic.