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

Abstract

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 k cat 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.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid

“…Tt CCO plays a crucial role in the coenzyme A-independent pathway for synthesizing vanillin. Tt CCO catalyzed the integration of molecular oxygen into 4-VP, facilitating the cleavage of specific carbon–carbon double bonds in the Fe-4 × His catalytic pocket . 4-VP is an electron-rich and redox-active molecule, providing the essential reducing equivalents for oxidative cleavage.…”

“…For example, the amidase-twisted substrate tunnel was engineered to be straightened, resulting in a major increase in enzyme activity . However, indiscriminately mutating all nonpolar hydrophobic residues near the substrate access channel with phenyl rings to smaller conformations is often unwise . These residues with phenyl rings might form π–π stacking intermolecular interactions with free substrates in solvents, thereby aiding in substrate recognition .…”

“…Enzyme engineering is considered an effective strategy for enhancing the rate-limiting CCO enzymes. − CCO is a nonheme iron enzyme whose conserved metal center consists of the 4 × His residues coordinating an iron cofactor. ,,− Utilizing its iron catalytic center, CCO integrates molecular oxygen into 4-VP, facilitating the cleavage of specific carbon–carbon double bonds. Moreover, the CCO’s catalytic pocket is connected to the protein’s surface via a channel, which plays a crucial role in substrate, product, and electron delivery. , Based on the above catalytic mechanism of CCO, the CCO’s directed evolution can be approached from substrate channel engineering and catalytic pocket engineering. For example, directed evolution guides channel expansion, allowing more substrate molecules to pass through .…”

Mid–Long Chain Dicarboxylic Acid Production via Systems Metabolic Engineering: Progress and Prospects

“…The formation of new carbon–carbon bonds within the carbon skeleton is a crucial step in compound derivatization, as it allows for the generation of complex organic compounds from simple precursors. Claisen condensation plays a significant role in the formation of carbon–carbon bonds . Thus, it is widely used in the biosynthesis of DCAs such as glutaric acid and adipic acid.…”

“…Claisen condensation plays a significant role in the formation of carbon− carbon bonds. 98 Thus, it is widely used in the biosynthesis of DCAs such as glutaric acid and adipic acid. Cheong et al 99 synthesized DCAs with different carbon chain lengths via the nondecarboxylative Claisen condensation reaction and a subsequent β-reduction reaction and carbon chain extension termination reaction.…”