How to complete the tautomerization and substrate-assisted activation prior to C–C bond fission by meta-cleavage product hydrolase LigY?

Abstract

LigY belongs to the amidohydrolase superfamily responsible for the C–C bond fission of meta-cleavage products (MCPs). LigY employs similar nucleophile-activation strategies like previously reported serine-dependent MCP hydrolases; however, the mechanisms of the tautomerization and hydrolysis are different. In this study, the quantum mechanics/molecular mechanics (QM/MM) approach was performed to elucidate the mechanism of MCP C–C bond fission by LigY. The binding (monodentate or bidentate) of the substrate to a zinc ion plays a significant role in the tautomerization. Both binding modes could complete the tautomerization and hydrolysis; however, the monodentate mode and five-coordination might be the most likely binding mode. For the bidentate binding mode, unlike the mechanism proposed based on experiments, Tyr190 acts as a “proton transfer” bridge to complete the tautomerization and indirectly activates the catalytic water. For the monodentate binding mode, interestingly, Tyr190/Arg234* (from the neighboring protomer B) act as a proton donor for the activated deprotonated water required to complete the tautomerization. A strong hydrogen bond is formed in this step to stabilize the water and prevent it from moving away from the substrate (C⁶-carbonyl), leading to the subsequent C–C bond fragmentation. Furthermore, Arg72 plays a crucial role in the monodentate binding of the substrate to Zn(II). Our work expanded the understanding of the mechanism of MCP C–C bond fragmentation by LigY, especially details of the tautomerization and substrate-assisted activation prior to C–C bond fission.