S. Monno et al., MUTATION OF HISTIDINE-373 TO LEUCINE IN CYTOCHROME-P450C17 CAUSES 17-ALPHA-HYDROXYLASE DEFICIENCY, The Journal of biological chemistry, 268(34), 1993, pp. 25811-25817
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 r
eaction-based site-directed mutagenesis, we prepared a DNA that encode
d the Leu373 mutant protein. COS-1 cells transfected with the mutant D
NA, despite having an RNA hybridizable to the P450c17 cDNA, did not sh
ow 17alpha-hydroxylase and 17,20-lyase activities. Also, the cells wer
e 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 e
xpressing the wild-type form of the modified enzyme showed an absorpti
on peak at 449 nm upon addition of carbon monoxide in the reduced stat
e and produced characteristic substrate-induced difference spectra, wh
ereas those from the cells expressing the mutant form did not show the
se spectral changes. The 17alpha-hydroxylase and 17,20-lyase activitie
s were observed only in E. coli cells expressing the wild-type enzyme.
These results show that the His373 --> Leu mutant does not incorporat
e the heme prosthetic group properly and suggest a critical role of Hi
s373 in heme binding.