Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450_(BM3) for Selective Hydroxylation of Large Substrates

Abstract

Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_(BM3) variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates.

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