Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3

Matthew W Peters, Peter Meinhold, Anton Glieder, Frances H. Arnold

Research output: Contribution to journalArticle

Abstract

Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min(-1)) and support thousands of product turnovers. The regio- and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.

Original languageEnglish
Pages (from-to)13442-50
Number of pages9
JournalJournal of the American Chemical Society
Volume125
Issue number44
DOIs
Publication statusPublished - 5 Nov 2003

Keywords

  • Alcohols
  • Alkanes
  • Bacillus megaterium
  • Bacterial Proteins
  • Cytochrome P-450 Enzyme System
  • Escherichia coli
  • Hydroxylation
  • Mixed Function Oxygenases
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • NADP
  • NADPH-Ferrihemoprotein Reductase
  • Protein Engineering
  • Stereoisomerism
  • Substrate Specificity
  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.

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