MUTATION OF HISTIDINE-373 TO LEUCINE IN CYTOCHROME-P450C17 CAUSES 17-ALPHA-HYDROXYLASE DEFICIENCY

We identified a new homozygous missense mutation His373 --> Leu in the CYP17 gene of two sisters with 17alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11beta-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His373 --> Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 Mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17alpha-hydroxylase and 17,20-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His373 --> Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding.