Cytochrome P450BM3 is a highly efficient heme enzyme whose native activity is limited to long-chain fatty acids. To overcome this limitation without genetic modification, we developed a mutation-free decoy molecule strategy, where inert compounds mimicking native substrates trigger oxygen activation. Using this method, wild-type P450BM3 catalyzed the oxidation of a broad range of non-native substrates, including benzene, propane, and methane, with initially purified enzyme preparations.
In a notable case, oxidation of methane to methanol was achieved at ambient temperature under 10 MPa methane pressure, with a turnover number of 4.0 ± 0.4. The decoy molecule 3CHPA-Pip-Phe was particularly effective not only in inducing catalytic activity but also in promoting crystallization of the enzyme–ligand complex, enabling detailed structural characterization. The high-resolution X-ray structure (1.54 Å) of the P450BM3–decoy complex revealed that the pipecolic acid moiety promotes a native-like "curved" substrate-binding conformation, while the cyclohexyl group seals the substrate channel, forming an enclosed active site pocket conducive to gas binding. Docking simulations indicated sufficient space for binding multiple methane molecules.
Importantly, this strategy was successfully extended to whole-cell systems, demonstrating its applicability in cellular environments without the need for mutagenesis. These results collectively establish a versatile and sustainable platform for programmable oxidation chemistry, with mechanistic insights supported by structure-guided analysis using the crystallization-enhancing decoy molecule N-abietoyl-L-tryptophan (AbiATrp). AbiATrp enabled rapid crystallization of the wild-type P450BM3 heme domain within 2 hours, and crystals obtained with AbiATrp served as seeds in cross-microseeding experiments, yielding diffraction-quality crystals within as little as 10 minutes. This approach allowed structural determination of previously intractable P450BM3 complexes and provided valuable snapshots of distinct stages within the catalytic cycle.