Design-informed construction of self-sufficient cytochrome P450 fusion enzymes for efficient avermectin oxidation

Cytochrome P450 enzymes hold immense potential as biocatalysts for oxidative reactions but often suffer from inefficient electron transfer due to their reliance on transient interactions with redox partners (RPs). To overcome this limitation, we constructed self-sufficient fusion enzymes by fusing the avermectin-oxidizing P450-Ema1-M212A to selected RPs. Linker sequences were primarily optimized experimentally, while computational modeling, including AlphaFold3-predicted multi-protein assemblies and molecular dynamics simulations, was used as an auxiliary tool to assess feasible linker lengths and interpret the structural basis by which optimized linker design enhances electron-transfer efficiency. Based on literature precedent and preliminary structural considerations, a series of fusion constructs with varying linker sequences and lengths was constructed. Molecular dynamics simulations suggested a structural rationale for why a linker length of 9 amino acids exhibited optimal enzymatic activity, as it stabilized a functional conformation, minimized structural fluctuations, and maintained a favorable distance between the heme and Fe₂S₂ clusters for efficient electron transfer. Experimental characterization confirmed that the F4 variant exhibited substantially increased activity over the non-fusion version. Kinetic analysis demonstrated a significantly enhanced k_cat/K_m ratio, indicating accelerated catalysis without compromising substrate binding. For the synthesis of 4″-oxo-avermectin, a key intermediate of emamectin, the F4 variant achieved 90% conversion within 8 h, far exceeding the performance of the non-fused system. This study establishes an experimentally driven, semi-rational framework for constructing self-sufficient P450 fusion enzymes, highlighting the synergy between computational prediction and protein engineering for developing powerful biocatalysts with high industrial application potential.