Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency

• Published in: The Journal of biological chemistry 1993-12, Vol.268 (34), p.25811-25817
• Main Authors: Monno, S, Ogawa, H, Date, T, Fujioka, M, Miller, W L, Kobayashi, M
• Format: Article
• Language: English
• Subjects: Adolescent; Adrenal Hyperplasia, Congenital; Adult; Alleles; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; binding; Biological and medical sciences; Blotting, Southern; Cell Line; Codon - genetics; cytochrome P450c17; deficiency; DNA - blood; DNA - isolation & purification; DNA Primers; Enzymes and enzyme inhibitors; Exons; Female; Fundamental and applied biological sciences. Psychology; heme; Histidine; Humans; Leucine; Leukocytes - enzymology; Male; man; Molecular Sequence Data; Mutagenesis, Site-Directed; mutation; Oxidoreductases; Point Mutation; Polymerase Chain Reaction; Restriction Mapping; role; steroid 17 alpha -monooxygenase; Steroid 17-alpha-Hydroxylase - genetics; Steroid 17-alpha-Hydroxylase - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74462-7

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.