Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3.
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.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Matthew W
MW
Peters
Peter
P
Meinhold
Anton
A
Glieder
Frances H
FH
Arnold
D000438:Alcohols / Q000138:chemical synthesis / Q000378:metabolism; D000473:Alkanes / Q000737:chemistry* / Q000378:metabolism; D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D015202:Protein Engineering / Q000379:methods; D013237:Stereoisomerism; D013379:Substrate Specificity
D000438:Alcohols; D000473:Alkanes; D001426:Bacterial Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases