Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.
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Monno S, Ogawa H, Date T, Fujioka M, Miller WL, Kobayashi M
Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.
J Biol Chem. 1993 Dec 5;268(34):25811-7.
- PubMed ID
- 8245018 [ View in PubMed]
- Abstract
We identified a new homozygous missense mutation His373-->Leu in the CYP17 gene of two sisters with 17 alpha-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 17 alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11 beta-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 17 alpha-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.