DFT-Based Elucidation of LigY-Catalyzed C-C Bond Cleavage Mechanism in Sphingobium sp. SYK-6

LigY is a Zn²⁺-dependent meta-cleavage product hydrolase involved in the bacterial degradation of lignin-derived aromatic compounds, catalyzing the hydrolysis of its native substrate DCHM-HOPDA, a key step in carbon cycling. Despite the similarity to LigJ, LigY lacks the canonical acidic residue for water activation, leaving its mechanism unresolved. Using quantum mechanical calculations, we examined two pathways: a substrate-assisted proton-relay pathway-I and an experimentally motivated pathway-II. Pathway-I is kinetically feasible, proceeding through three steps with a rate-limiting barrier of 19.3 kcal/mol, consistent with experimental turnover rates. Tyr190 acts as a bifunctional acid-base, His29 mediates proton relay, and Glu282 transiently stabilizes the gem-diol intermediate, while Zn²⁺ anchors and polarizes the substrate for efficient catalysis. In contrast, pathway-II is kinetically inaccessible (40.2 kcal/mol). Calculations rationalize mutagenesis and substrate specificity data, highlighting the roles of Tyr190, Glu282, Arg72, and Arg234. These results clarify LigY's catalytic mechanism and emphasize the importance of coordinated proton-shuttling networks in Zn²⁺-dependent hydrolases, providing insights for related enzymes and enzyme engineering.