Rational Design of the Substrate Tunnel of β-Ketothiolase Reveals a Local Cationic Domain Modulated Rule that Improves the Efficiency of Claisen Condensation

Formation of carbon–carbon bonds via Claisen condensation catalyzed by thiolase generates a diverse range of carbon frameworks in biosynthetic chemistry. During catalysis, the substrate tunnel of thiolases plays an important role in the binding and condensation of substrates, directly affecting enzyme activity. Therefore, rational engineering of the substrate tunnel is a promising approach for enhancing enzyme performance. However, the lack of a general engineering rule hinders rational engineering of the substrate tunnel. In this study, we reported the crystal structure of Tfu_0875, a thermostable β-ketothiolase from Thermobifida fusca belonging to the thiolase superfamily. The enzyme had a Cys–His–Cys catalytic triad and a narrow substrate tunnel mainly built from the cationic domain, the adenine-binding pocket, and the pantetheine loop. Focusing on the substrate tunnel, mutant candidates with increased kcat were predicted by the deep learning approach. The best mutation in the cationic domain (L163H) exhibited a 313% increase in enzyme activity compared with the wild-type enzyme. Molecular dynamics simulations revealed a local cationic domain modulated rule (LCDMR): mutating the nonconserved residue at the junction between the loop region and the α5 region in the sandwich topology to histidine significantly improved enzyme activity by increasing the reaction space and interaction with the substrate or unstable intermediate. The LCDMR has general applicability in rational engineering of the substrate tunnel to enhance the enzyme activity of other β-ketothiolases.