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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alcohols / chemical synthesis
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Alcohols / metabolism
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Alkanes / chemistry*
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Alkanes / metabolism
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Bacillus megaterium / enzymology
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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Cytochrome P-450 Enzyme System / chemistry*
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Cytochrome P-450 Enzyme System / genetics*
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Cytochrome P-450 Enzyme System / metabolism
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Hydroxylation
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Mixed Function Oxygenases / chemistry*
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Mixed Function Oxygenases / genetics*
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Mixed Function Oxygenases / metabolism
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Models, Molecular
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Mutagenesis, Site-Directed
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NADP / chemistry
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NADP / metabolism
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NADPH-Ferrihemoprotein Reductase
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Protein Engineering / methods
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Stereoisomerism
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Substrate Specificity
Substances
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Alcohols
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Alkanes
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Bacterial Proteins
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NADP
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Cytochrome P-450 Enzyme System
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Mixed Function Oxygenases
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NADPH-Ferrihemoprotein Reductase
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flavocytochrome P450 BM3 monoxygenases