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.

• Publication year

  1993

• Venue

  Journal of Biological Chemistry

• Publication date

  1993-02-25

• Fields of study

  Medicine, Chemistry, Engineering

• Identifiers
  DOI 10.1016/s0021-9258(18)53630-9PMID 8440727
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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