A self-sufficient peroxide-driven hydroxylation biocatalyst.

Controlled, selective oxidations of unactivated C H bonds are among the most desired transformations in catalysis. Limitations to chemical oxidation catalysts include low turnover numbers, low or no regioselectivity, poor reaction specificity, their use of environmentally harmful components (for example, heavy metals or halogens), or their requirements for harsh, expensive reaction conditions. Many oxygenase enzymes catalyze the insertion of oxygen into unactivated C H bonds and are superior to synthetic catalysts with regard to selectivity and turnover rate. Enzymes have found numerous industrial applications, and the use of enzymes in industrial biocatalysis is expected to grow significantly. One limitation to the application of oxygenases is their requirement for an expensive cofactor such as NAD(P)H, which precludes their use in vitro without addition of a coupled, cofactor-regenerating system. The need for additional proteins to transfer electrons from the cofactor to the oxygenase presents a further complication. Here we describe a self-sufficient P450 BM-3 variant which utilizes hydrogen peroxide (H2O2) to catalyze hydroxylation and epoxidation at high rates. The cytochromes P450 are heme-containing oxygenases which collectively catalyze a variety of oxidations on a diverse array of substrates. The natural P450 catalytic cycle utilizes two electrons from NAD(P)H to activate dioxygen, although P450s are also capable of utilizing peroxides as a source of oxygen through a peroxide “shunt” pathway. In principle, this “peroxygenase” activity offers an opportunity to employ cellfree P450 catalysis without requiring NAD(P)H regeneration, additional proteins, or dioxygen, and eliminates rate-limiting electron-transfer steps. In practice, this non-natural pathway is too inefficient for any practical application. The soluble P450 BM-3 enzyme catalyzes the hydroxylation of sub-terminal C H bonds of medium chain (C12–C18) fatty acids and amides with initial rates exceeding 3000 min . The activities of P450 BM-3 and its corresponding F87A mutant in reactions driven by H2O2 were recently characterized. Whereas the wild-type enzyme is inactivated after only a few turnovers, the F87A mutant supports low levels of peroxygenase activity (ca. 70 total turnovers). In a previous study we demonstrated that directed evolution could increase the rate of peroxide-driven naphthalene hydroxylation by cytochrome P450cam. Since then, our efforts have focused on improving the peroxygenase activity of the more active P450 BM-3 as a step towards engineering a “biomimetic” hydroxylation catalyst. The P450 BM-3 heme domain (BMP) supports peroxygenase activity and is more thermostable than the fulllength P450 BM-3 protein (unpublished data). Removal of the reductase domain also results in an approximate fourfold increased molar expression (ca. 70 mgL 1 in shake-flask cultures). We used BMP mutant F87A (HF87A) as the starting point (parent) to increase catalyst performance by sequential rounds of random mutagenesis and screening for H2O2-driven hydroxylation of 12-p-nitrophenoxycarboxylic acid (12-pNCA). Experimental details and a description of the evolutionary path are available in the Supporting Information. Fifth-generation mutant “21B3” is nearly 20-fold more active than HF87A in 10 mm H2O2 using 12-pNCA as the substrate. Figure 1 shows the activities of HF87A and 21B3