Structural snapshot of a glycoside hydrolase family 8 endo-β-1,4-glucanase capturing the state after cleavage of the scissile bond.

Abstract

Bacterial cellulose (BC), which is produced by bacteria, is a biodegradable and biocompatible natural resource. Because of its remarkable physicochemical properties, BC has attracted attention for the development and manufacture of biomedical and industrial materials. In the BC production system, the enzyme endo-β-1,4-glucanase, which belongs to glycoside hydrolase family 8 (GH8), acts as a cleaner by trimming disordered cellulose fibers to produce high-quality BC. Understanding the molecular mechanism of the endo-β-1,4-glucanase would help in developing a reasonable biosynthesis of BC. Nevertheless, all of the steps in the reaction of this endo-β-1,4-glucanase are not clear. This study confirms the BC hydrolytic activity of the endo-β-1,4-glucanase from the BC-producing bacterium Enterobacter sp. CJF-002 (EbBcsZ) and reports crystal structures of EbBcsZ. Unlike in previously reported GH8 endo-β-1,4-glucanase structures, here the base catalyst was mutated (D242A) and the structure of this mutant bound to cellooligosaccharide [EbBcsZ(D242A)CPT] was analyzed. The EbBcsZ(D242A)CPT structure showed two cellooligosaccharides individually bound to the plus and minus subsites of EbBcsZ. The glucosyl unit in subsite -1 presented a distorted 5S1 conformation, a novel snapshot of a state immediately after scissile-bond cleavage. In combination with previous studies, the reaction process of endo-β-1,4-glucanase is described and the β-1,4-glucan-trimming mechanism of EbBcsZ is proposed. The EbBcsZ(D242A)CPT structure also showed an additional β-1,4-glucan binding site on the EbBcsZ surface, which may help to accept the substrate.