title: Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.
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.
authors.affiliation: Department of Internal Medicine, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Monno
authors.forename: H
authors.initials: H
authors.lastname: Ogawa
authors.forename: T
authors.initials: T
authors.lastname: Date
authors.forename: M
authors.initials: M
authors.lastname: Fujioka
authors.forename: W L
authors.initials: WL
authors.lastname: Miller
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital; D000328:Adult; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003062:Codon / Q000235:genetics; D004247:DNA / Q000097:blood / Q000302:isolation & purification; D017931:DNA Primers; D005091:Exons; D005260:Female; D006639:Histidine; D006801:Humans; D007930:Leucine; D007962:Leukocytes / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D003062:Codon; D017931:DNA Primers; D006639:Histidine; D004247:DNA; D013254:Steroid 17-alpha-Hydroxylase; D007930:Leucine