Molecular basis for a functionally unique cytochrome P450IIB1 variant

K. M. Kedzie, C. A. Balfour, G. Y. Escobar, S. W. Grimm, Y. A. He, D. J. Pepperl, J. W. Regan, J. C. Stevens, J. R. Halpert

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108 Scopus citations

Abstract

Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16β-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16β-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16β-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16α-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16β-OH:16α-OH = 1.4) is thus distinct from that (16β-OH:16α-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478 →Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.

Original languageEnglish (US)
Pages (from-to)22515-22521
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number33
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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