Combinatorial alanine substitution enables rapid optimization of cytochrome P450BM3 for selective hydroxylation of large substrates

Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450(BM3) (BM3) variant was used to generate BM3 variants with activity on large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible. This approach could be generally useful for improving the activity of enzymes that show only limited activity on larger substrates.