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

Selective hydroxylation of C H bonds in organic compounds provides an efficient means to access valuable drug metabolites, natural product derivatives, and other fine chemicals. While chemical methods to accomplish this transformation have improved, these generally require the presence of directing groups or electronic properties inherent to certain substrate classes for the desired transformations to occur at all or with useful regioselectivity. Enzymes are capable of avoiding this limitation by employing potent H-abstraction mechanisms with selectivity imposed by specific substrate binding. Furthermore, systematic optimization of these catalysts by directed evolution has been demonstrated extensively and constitutes a powerful advantage of these systems over small molecule catalysts. Members of the cytochrome P450 monooxygenase superfamily are remarkable examples of such catalysts. These enzymes utilize a cysteine-bound heme cofactor to catalyze a wide range of oxidative transformations including hydroxylation and epoxidation. Cytochrome P450BM3 (BM3) from Bacillus megaterium possesses a number of features that make it particularly attractive for applications in chemical synthesis. For example, the heme domain in which hydroxylation occurs and the diflavin reductase domains (FMN and FAD) that contribute electrons for oxygen activation in the heme domain are fused in a single polypeptide chain; this improves the rate and operational simplicity of BM3-catalyzed reactions. Indeed, BM3 catalyzes the subterminal hydroxylation of C12–C18 fatty acids, its natural substrates, at rates of thousands of turnovers per minute, making it one of the most active hydroxylases known. BM3 is soluble, readily over-expressed in a variety of heterologous hosts, and requires only atmospheric oxygen and a supply of nicotinamide adenine dinucleotide phosphate (NADPH) for hydroxylation activity. These properties have led our groups and others to expand the substrate scope of this enzyme with the goal of creating efficient enzyme and wholecell catalysts for a variety of oxidative transformations. The development of BM3 variants that catalyze regioselective demethylation or demethoxymethylation of methylated or methoxymethylated monosaccharides was recently reported by our groups. While a chemoenzymatic method employing these enzymes provided a convenient means to access otherwise difficult-to-synthesize monosaccharide derivatives, it also highlighted some limitations of existing BM3 variants. For example, while MOM-protected pentoses were compatible with these enzymes, MOM-protected hexoses were deprotected to only a minor extent (Scheme 1). This same limitation was