Mechanistic Insights into Lysine Cyclodeaminase Catalysis.

Lysine cyclodeaminase (LCD) catalyzes the conversion of l-lysine into l-pipecolic acid, a key building block for food additives and pharmaceutical intermediates. Despite its industrial relevance, LCD displays a narrow substrate scope, efficiently converting l-lysine, while bulkier derivatives such as l-lysine ethyl ester fail to undergo productive biotransformation. To elucidate the molecular origin of this selectivity and define the catalytic mechanism, we combined molecular docking, substrate tunnel engineering, classical molecular dynamics, well-tempered metadynamics simulations, and experimental validation. Computational analyses show that both l-lysine and l-lysine ethyl ester can access and bind within the LCD active site, and tunnel engineering produced LCD variants (I61V-I94V-D236C and I61V-I94V-E264T) with improved tunnel properties. However, experimental assays demonstrated that these variants did not acquire catalytic activity toward l-lysine ethyl ester. Mechanistic simulations reveal that the proposed l-lysine iminium intermediate consistently adopts low-energy, cyclization-competent conformations in which the nucleophilic Nε and reactive Cα atoms achieve near-attack geometries. In contrast, the proposed l-lysine ethyl ester iminium intermediate populates higher-energy states with misaligned geometries and kinetically trapped conformations, suggesting difficult cyclization despite successful binding. These findings suggest that steric and dynamic constraintsrather than substrate accesscould impact the catalysis of esterified substrates. This work establishes a mechanistic framework linking enzyme dynamics, substrate recognition, and catalytic efficiency, providing a foundation for rational LCD engineering aimed at expanding substrate scope and guiding future industrial applications.