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

摘要

LigY belongs to the amidohydrolase superfamily responsible for the C-C bond fission ofmeta-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 (C-6-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.