Pitfalls in characterizing P450c17 mutations associated with isolated 17,20-lyase deficiency

The cytochrome P450c17 enzyme system performs both the 17 alpha -hydroxylas e and 17,20-lyase reactions in the human adrenal glands and gonads. This 17 ,20-lyase activity is required for the biosynthesis of dehydroepiandrostero ne, the C-19 precursor of sex steroids. Considerable evidence supports the idea that the 17,20-lyase activity of this system is particularly sensitive to alterations in the interactions between P450c17 and its cofactor protei ns P450-oxidoreductase and cytochrome b(5). We have described two patients with the clinical phenotype of isolated 17,20-lyase deficiency in whom sing le amino acid replacement mutations in the redox partner binding site of P4 50c17 (R347H and R358Q) selectively ablate 17,20-lyase activity while prese rving most 17 alpha -hydroxylase activity. We have shown by computer modeli ng and detailed biochemical studies that mutations R347H and R358Q impair t he interactions of P450c17 with P450-oxidoreductase and cytochrome b(5) (re dox partners). Another mutation reported to cause isolated 17,20-lyase defi ciency (F417C) does not map within the redox partner binding site, but migh t nonetheless alter the interaction of the mutant protein with redox partne rs. To study the interaction of the F417C mutation with P450 oxidoreductase and cytochrome b(5), we expressed the cDNA for this protein in yeast micro somes, a heterologous expression system in which the composition of redox p artner proteins can be varied systematically. Although the full-length prot ein was expressed in quantities comparable to those of wild-type P450c17 in this system, the F417C mutation did not form a classical P450 difference s pectrum and was devoid of both 17 alpha -hydroxylase and 17,20-lyase activi ties. To ensure that this result was not unique to the yeast expression sys tem, we also expressed wild-type P450c17 and the F417C mutation in COS-7 ce lls, and we again found that the F417C mutation was expressed, but was not active. To conclusively demonstrate that a particular mutation in P450c17 c auses isolated 17,20-lyase deficiency, accurate enzymatic studies of the mu tant protein must reproducibly show activities consistent with the diagnosi s. Mutations R347H and R358Q are the only two such mutations found in human s proven to cause isolated 17,20-lyase deficiency.