Sigmatropic rearrangements, while rare in biology, offer opportunities for the efficient and selective synthesis of complex chemical motifs. A “P411” serine-ligated variant of cytochrome P450_BM3 has been engineered to initiate a sulfimidation/[2,3]-sigmatropic rearrangement sequence in whole E. coli cells, a non-natural function for any enzyme, providing access to enantioenriched, protected allylic amines. Five mutations in the enzyme substantially enhance its activity toward this new function, demonstrating the evolvability of the catalyst toward challenging nitrene transfer reactions. The evolved catalyst additionally performs the highly enantioselective imidation of non-allylic sulfides.

Sigmatropic rearrangements, while rare in biology, offer opportunities for the efficient and selective synthesis of complex chemical motifs. A “P411” serine-ligated variant of cytochrome P450_BM3 has been engineered to initiate a sulfimidation/[2,3]-sigmatropic rearrangement sequence in whole E. coli cells, a non-natural function for any enzyme, providing access to enantioenriched, protected allylic amines. Five mutations in the enzyme substantially enhance its activity toward this new function, demonstrating the evolvability of the catalyst toward challenging nitrene transfer reactions. The evolved catalyst additionally performs the highly enantioselective imidation of non-allylic sulfides.

Enzymes capable of catalyzing non-natural reactions have the potential to alter the way relevant molecules are prepared on-scale. Efforts to this end have largely focused on combining non-natural cofactors with proteins lacking catalytic function to obtain non-natural reactivity. An alternative approach is to utilize a native cofactor to catalyze non-natural reactions. Recently, our group demonstrated that heme-containing cytochrome P450s are able to catalyze the highly selective cyclopropanation of alkenes. Superior activity was observed upon changing the axial cysteine to serine (“P411”). Mutation at the conserved axial ligand has enabled P450s to catalyze other non-natural reactions such as NH insertion and CH amination.

Recent work has shown that engineered variants of cytochrome P450_BM3 (CYP102A1) efficiently catalyze non-natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range of natural P450 enzymes, we set out to explore if this diversity could be leveraged to generate a broad panel of new catalysts for olefin cyclopropanation (i.e., carbene transfer). Here, we took a step towards this goal by characterizing the carbene transfer activities of four new wild-type P450s that have different native substrates. All four were active and exhibited a range of product selectivities in the model reaction: cyclopropanation of styrene by using ethyl diazoacetate (EDA). Previous work on P450_BM3 demonstrated that mutation of the axial coordinating cysteine, universally conserved among P450 enzymes, to a serine residue, increased activity for this non-natural reaction. The equivalent mutation in the selected P450s was found to activate carbene transfer chemistry both in vitro and in vivo. Furthermore, serum albumins complexed with hemin were also found to be efficient in vitro cyclopropanation catalysts.

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB′₁ helix of the protein to seal the binding pocket.

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity.

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity.

Simple and universal methods for the preparation of human drug metabolites are required to produce quantities sufficient for their characterization and toxicity testing. Synthetic chemistry lacks general catalysts for selective oxidation of unactivated CH bonds, a transformation that plays a key role in metabolism; bioconversions using P450 enzymes have emerged as a powerful alternative. Variants of P450_BM3 from Bacillus megaterium act on diverse substrates, including drugs. Acidic substrates, such as the compounds metabolized by CYP2C9, which is one of three main hepatic human P450s, are not accepted by P450_BM3 variants engineered to date. Herein, we report bacterial mimics of CYP2C9, which are active on two widely administered drugs, naproxen and ibuprofen, that are CYP2C9 substrates in vivo. These P450_BM3 variants can also act on desmethylnaproxen, the human metabolite of naproxen, and convert it to the 1,4-naphthoquinone derivative. We analyzed the crystal structure of the heme domain of an early intermediate in the directed-evolution experiment. The active site mutation, L75R, which initially conferred activity on charged substrates, dramatically increased structural flexibility in the B′-helix. This increased flexibility, which was accompanied by a dramatic decrease in enzyme stability, may contribute to the variant’s ability to accept a broader range of substrates.

Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant.