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CH activation
directed evolution
enantioselectivity
oxidation
P450 enzymes
[
Abstract:
Directed evolution of the monooxygenase P450-BM3 utilizing
iterative saturation mutagenesis at and near the binding site
enables a high degree of both regio- and enantioselectivity in
the oxidative hydroxylation of cyclohexene-1-carboxylic acid
methyl ester. Wild-type P450-BM3 is 84% regioselective for the
allylic 3-position with 34% enantioselectivity in favor of the R
alcohol. Mutants enabling R selectivity (>95% ee) or S selectivity
(>95% ee) were evolved, while reducing other oxidation
products and thus maximizing regioselectivity to >93%. Control
of the substrate-to-enzyme ratio is necessary for obtaining
optimal and reproducible enantioselectivities, an observation
which is important in future protein engineering of these
mono-oxygenases. An E. coli strain capable of NADPH regeneration
was also engineered, simplifying directed evolution of
P450 enzymes in general. These synthetic results set the stage
for subsequent stereoselective and stereospecific chemical
transformations to form more complex compounds, thereby
illustrating the viability of combining genetically altered enzymes
as catalysts in organic chemistry with traditional chemical
methods.
