The catalytic activity of cytochrome P450cam towards styrene oxidation is increased by site-specific mutagenesis

The styrene oxidation activity of cytochrome P450cam has been greatly improved by rational protein engineering. Compared to the wild-type enzyme, the active-site mutants Y96A and Y96F bound styrene more tightly, consumed NADH more rapidly, and were more efficient at utilising reducing equivalents fo...

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Bibliographic Details
Published in:FEBS letters 1997-03, Vol.405 (2), p.153-156
Main Authors: Nickerson, Darren P, Harford-Cross, Charles F, Fulcher, Sarah R, Wong, Luet-Lok
Format: Article
Language:English
Subjects:
Binding Sites - genetics
Camphor 5-Monooxygenase - genetics
Camphor 5-Monooxygenase - metabolism
Epoxidation
Monooxygenase
Mutagenesis
Mutagenesis, Site-Directed
Mutation
Oxidation-Reduction
P450
Protein Engineering
Spectrophotometry
Stereoselectivity
Styrene
Styrenes - metabolism
Substrate Specificity
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Summary:The styrene oxidation activity of cytochrome P450cam has been greatly improved by rational protein engineering. Compared to the wild-type enzyme, the active-site mutants Y96A and Y96F bound styrene more tightly, consumed NADH more rapidly, and were more efficient at utilising reducing equivalents for product formation. Styrene oxide formation rates were enhanced 9-fold in the Y96A mutant relative to wild-type, and 25-fold in the Y96F mutant, thus demonstrating the effectiveness of active-site redesign in improving the activity of a haem monooxygenase towards an unnatural substrate. ©1997 Federation of European Biochemical Societies.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(97)00174-9