Abstract
The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacillus megaterium / chemistry
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Bacillus megaterium / enzymology*
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Bacillus megaterium / genetics
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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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Epoxy Compounds / metabolism*
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Industrial Microbiology
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Models, Molecular
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Mutagenesis, Site-Directed*
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Mutant Proteins / chemistry
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Mutant Proteins / genetics
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Mutant Proteins / metabolism
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Mutation
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NADPH-Ferrihemoprotein Reductase / chemistry
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NADPH-Ferrihemoprotein Reductase / genetics
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NADPH-Ferrihemoprotein Reductase / metabolism*
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Protein Binding
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Protein Conformation
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Styrene / metabolism*
Substances
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Bacterial Proteins
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Epoxy Compounds
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Mutant Proteins
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Styrene
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Cytochrome P-450 Enzyme System
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NADPH-Ferrihemoprotein Reductase
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flavocytochrome P450 BM3 monoxygenases