A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction.

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues.