ESTEEM Strategy: Revolutionizing Enzymatic Catalysis through the Evolutionary Gateway of Enzyme Tunnels

Elucidating enzyme behavior and engineering highly efficient biocatalysts remain fundamental challenges in biocatalysis. Despite advances in enzyme engineering, significant optimization opportunities persist. Herein, we introduce the Evolutionary-Structural Tuning of Enzyme Efficiency Modules (ESTEEM) strategy, which integrates evolutionary principles with structural scaffold analysis to accelerate enzyme design. Using cytochrome P450 monooxygenase (P450s)─enzymes with deeply buried active sites and intricate substrate tunnels─as models, we identified key nonconserved residues (I67 and Q83) within the substrate tunnel. These distal positions critically influence tunnel architecture, and their mutation dramatically enhanced catalytic efficiency. The optimal double-variant exhibited a 106-fold increase in total turnover number without compromising stability while simultaneously broadening the substrate scope. Through adaptive steered molecular dynamics and hydrogen–deuterium exchange mass spectrometry, we uncovered a tunnel-mediated tuning mechanism: activity differences between variants and wild type were governed by a rigidity-flexibility balance in the scaffold, which modulates catalytic efficiency by stabilizing tunnel geometry. Experimental validation and retrospective analysis confirmed the robustness of the ESTEEM strategy. This study establishes how tunnel mutations stabilize substrate transport pathways to enhance catalysis, providing a framework for engineering enzymes specialized activity toward substrates requiring deeply recessed active sites─advancing applications in biocatalysis and synthetic biology.