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

ABSTRACT: Human CYP17 (17alpha-hydroxylase-17,20-lyase; also cytochrome P450c17 or cytochrome P450(17alpha)) 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, 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 46XY male patients. By using site-directed mutagenesis we show here that another mutation in human CYP17, Arg(449)-->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 b(5), 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 Arg(347), Arg(358), Arg(449)) are vital for the functional interaction of CYP17 with cytochrome b(5) and that the loss of any one of these cationic charges is catastrophic.

SUBMITTER: Lee-Robichaud P

PROVIDER: S-EPMC1220466 | biostudies-other | 1999 Sep

REPOSITORIES: biostudies-other

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Project description:Human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17alpha-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1-98, followed by a membrane insertable C-terminal tail, residues 99-133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (core-tail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signal-core) and the latter containing the C-terminal tail of the native rat protein (signal-core-tail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and core-tail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signal-core-tail was 55% as efficient. The core and signal-core constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.

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

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