Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies.
Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.
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Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
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Halpert
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D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002701:Chloramphenicol / Q000378:metabolism / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D000735:Androstenedione; D002701:Chloramphenicol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase