The role of cytochrome 2B1 substrate recognition site residues 115, 294, 297, 298, and 362 in the oxidation of steroids and 7-alkoxycoumarins.
At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s. The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation. Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile. In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16alpha-hydroxytestosterone:16beta-hydroxytestosterone, with the most dramatic alteration being displayed by A298V. Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates. Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites. The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families.
Domanski
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TL
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555, USA. tadomans@utmb.edu
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Scott
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Wang
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Halpert
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D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003374:Coumarins / Q000737:chemistry* / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006900:Hydroxylation; D008958:Models, Molecular; D009154:Mutation; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013256:Steroids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism
D003374:Coumarins; D016877:Oxidants; D011993:Recombinant Fusion Proteins; D013256:Steroids; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C022724:testosterone 7-alpha-hydroxylase, hamster