A novel ring-shaped reaction pathway with interconvertible intermediates in chitinase A as revealed by QM/MM simulation combined with a one-dimensional projection technique.

Substrate-assisted catalysis (SAC), a mechanism of chitin hydrolysis by chitinases belonging to the glycoside hydrolase family 18 (GH18), has been studied experimentally and theoretically for several decades. However, the detailed reaction mechanism in chitinase A (ChiA) remains unclear at the atomic level. In this study, we investigated glycosylation, the first step of SAC, of ChiA obtained from Serratia marcescens (SmChiA), using QM/MM simulations combined with a one-dimensional projection (ODP) technique, which enabled us to explore the multi-dimensional free energy surface efficiently. The results showed that the reaction proceeds via a novel ring-shaped concerted reaction pathway with interconvertible intermediates, viz. oxazolinium ion and oxazoline, which have not been fully identified in previous studies. We also compared this chitin hydrolysis mechanism in SmChiA with that in SmChiB reported previously. The computational protocol developed in this study could also be applicable for elucidating complicated reaction mechanisms in other enzymes.