Molecular basis for a functionally unique cytochrome P450IIB1 variant

• Published in: The Journal of biological chemistry 1991-11, Vol.266 (33), p.22515-22521
• Main Authors: Kedzie, K M, Balfour, C A, Escobar, G Y, Grimm, S W, He, Y A, Pepperl, D J, Regan, J W, Stevens, J C, Halpert, J R
• Format: Article
• Language: English
• Subjects: alanine; Alleles; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cloning, Molecular; Cytochrome P-450 CYP2B1; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450IIB1; DNA - genetics; DNA - isolation & purification; endoplasmic reticulum; Fundamental and applied biological sciences. Psychology; Genetic Variation; Hemoproteins; Kinetics; Liver - enzymology; Male; Metalloproteins; Microsomes, Liver - drug effects; Microsomes, Liver - enzymology; Oxidoreductases - genetics; Oxidoreductases - isolation & purification; Oxidoreductases - metabolism; Phenobarbital - pharmacology; Plasmids; Proteins; Rats; Rats, Inbred Lew; Rats, Inbred Strains; Rats, Inbred WF; Rats, Inbred WKY; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA - genetics; RNA - isolation & purification; Sequence Homology, Nucleic Acid; Species Specificity; Steroid 16-alpha-Hydroxylase; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)54602-0

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 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-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 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-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 (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-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.