Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-β-1,3-glucanase on β1-3/1-6-glucan

The glycoside hydrolase family 55 (GH55) includes inverting exo-beta-1,3-glucosidases and endo-beta-1,3-glucanases, acting on laminarin, which is a beta 1-3/1-6-glucan consisting of a beta 1-3/1-6-linked main chain and beta 1-6-linked branches. Despite their different modes of action toward laminarin, endo-beta-1,3-glucanases share with exo-beta-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investi-gated the mechanism of endo-type action on laminarin by GH55 endo-beta-1,3-glucanase MnLam55A, identified from Micro-dochium nivale. MnLam55A, like other endo-beta-1,3-glucanases, degraded internal beta-D-glucosidic linkages of laminarin, producing more reducing sugars than the sum of D-glucose and gentiooli-gosaccharides detected. beta 1-3-Glucans lacking beta 1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal beta 1-3-linkage of the laminar-ioligosaccharide moiety at the reducing end side of the main chain beta 1-6-linkage. MnLam55A liberates D-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to lami-narioligosaccharides (<= 4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that beta-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 angstrom resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-beta-1,3-glucosidases. However, MnLam55A pos-sesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydro-lysis of internal linkages in laminarin is presumably common to GH55 endo-beta-1,3-glucanases.

Automated summary

The mechanism of endo-type action on laminarin by GH55 endo-beta-1,3-glucanase MnLam55A was investigated, revealing its preference for cleaving the nonreducing terminal beta 1-3-linkage of the laminar-ioligosaccharide moiety. The crystal structure of MnLam55A showed similarities to exo-beta-1,3-glucosidases, but possessed an extended substrate-binding cleft.