Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

TLDR: The goal was to engineer a P450BM3 variant with nativelike activity and coupling efficiency towards a structurally challenging, nonnative substrate (propane) and evaluate the impact of these features on performance in preparative-scale biotransformations.

Abstract: Cytochrome P450 enzymes (P450s) are exceptional oxygenating catalysts with enormous potential in drug discovery, chemical synthesis, bioremediation, and biotechnology. Compared to their natural counterparts, however, engineered P450s often exhibit poor catalytic and cofactor coupling efficiencies. Obtaining native-like catalytic proficiencies is a mandatory first step towards utilizing the power of these versatile oxygenases in chemical synthesis. Cytochrome P450BM3 (119 kDa, B. megaterium) catalyzes the subterminal hydroxylation of long-chain (C12–C20) fatty acids. Its high activity and catalytic self-sufficiency (heme and diflavin reductase domains are fused in a single polypeptide chain) 5] make P450BM3 an excellent platform for biocatalysis. However, despite numerous reports of the heme domain being engineered to accept nonnative substrates, including short-chain fatty acids, aromatic compounds, alkanes, and alkenes, reports of preparative-scale applications of P450BM3 remain scarce. [9] P450BM3 function is finely regulated through conformational rearrangements in the heme and reductase domains and possibly also through hinged domain motions. 10] Hydroxylation of fatty acids occurs almost fully coupled to cofactor (NADPH) utilization (93–96% depending on the substrate). In the presence of nonnative substrates or when amino acid substitutions are introduced, the mechanisms controlling efficient catalysis in P450s are disrupted, leading to the formation of reactive oxygen species and rapid enzyme inactivation. High coupling efficiencies on substrates whose physicochemical properties are substantially different from the native substrates have not been achieved, and coupling efficiencies ranging from less than 1% to 30– 40% are typical. 8] Strategies for addressing this “coupling problem” are needed in order to take engineered P450s to larger-scale applications. Selective hydroxylation of short alkanes is a long-standing problem, for which no practical catalysts are available. In an effort to produce P450BM3-based biocatalysts for selective hydroxylation of small alkanes, we previously engineered this enzyme to accept propane and ethane (35E11 variant). Despite greater than 5000 total turnover (TTN) supported in vitro, the utility of this catalyst remained limited because of its poor in vivo performance (see below), which was mostly due to the low efficiencies for coupling the product formation to cofactor consumption (17.4% for propane and 0.01% for ethane oxidation). Our goal was to engineer a P450BM3 variant with nativelike activity and coupling efficiency towards a structurally challenging, nonnative substrate (propane) and evaluate the impact of these features on performance in preparative-scale biotransformations. To this end, we used a domain-based protein-engineering strategy, in which the heme, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) domains of the 35E11 variant were evolved separately in the context of the holoenzyme, and beneficial mutations were recombined in a final step (Figure 1). Previous work suggested that mutations in the reductase and linker regions can affect catalytic properties. However, no systematic engineering efforts had been undertaken to engineer the complete 1048 amino acid holoenzyme. Holoenzyme libraries outlined in Figure 1 were created using random, saturation, and site-directed mutagenesis and