Engineering of cytochrome P450 2B1 specificity. Conversion of an androgen 16 beta-hydroxylase to a 15 alpha-hydroxylase.
Six site-directed mutants of cytochrome P450 2B1 were constructed, and function was evaluated in COS cell microsomes by monitoring testosterone and androstenedione hydroxylation and inactivation by chloramphenicol. Mutants Ile-114-->Val and Ile-114-->Ala exhibited marked decreases in androgen 16 beta-OH:16 alpha-OH ratios and increases in 15 alpha-OH:16-OH ratios. Since substitution of Gly-478 with Ala or Ser reduces 16 beta-hydroxylation in favor of 15 alpha-hydroxylation, four double mutants containing Val or Ala at position 114 and Ala or Ser at position 478 were examined. For any given residue at position 114 (Ile, Val, or Ala), the 15 alpha-OH:16-OH ratio increased as residue 478 was changed from Gly to Ala to Ser, and for any residue at position 478, this ratio increased as residue 114 was changed from Ile to Val to Ala. As a consequence, the Ile-114-->Ala, Gly-478-->Ser mutant displayed an approximately 1000-fold higher androgen 15 alpha-OH:16-OH ratio compared with the parental enzyme and functionally resembles mouse P450 2A4 much more closely than P450 2B1. All three mutants with Val at position 114 retained susceptibility to inactivation by chloramphenicol, whereas inactivation was suppressed by Ala at this position. The results suggest the feasibility of an empirical approach to P450 engineering involving the appropriate combination of residues at a few critical sites to confer new regio- and stereoselectivity with retention of overall monooxygenase activity.
Halpert
J R
JR
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
He
Y A
YA
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002701:Chloramphenicol / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004277:DNA, Single-Stranded; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012377:Rodentia; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D013739:Testosterone / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D004277:DNA, Single-Stranded; D007527:Isoenzymes; D013739:Testosterone; D000735:Androstenedione; D002701:Chloramphenicol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C460223:Cyp2a4 protein, mouse; D000072467:Cytochrome P450 Family 2