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.
Peters
Matthew W
MW
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Meinhold
Peter
P
Glieder
Anton
A
Arnold
Frances H
FH
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