Structural and biochemical basis for activity of Aspergillus nidulans α-1,3-glucanases from glycoside hydrolase family 71.

The microbial polysaccharide α-1,3-glucan is an important component of fungal cell walls and dental plaque biofilms, contributing to microbial virulence and biofilm resilience. Glycoside hydrolase family 71 (GH71) includes α-1,3-glucan degrading enzymes which could be exploited for biotechnological applications; however, the family is presently poorly understood. To increase our understanding of GH71, we have performed a phylogenetic analysis of the family and detailed biochemical analysis of two of the five GH71 enzymes encoded by Aspergillus nidulans (AnGH71B and -C). Both are active on soluble α-1,3-glucooligosaccharides but surprisingly only minimally on water-insoluble α-1,3-glucan. Assays on intact and milled A. nidulans biomass indicate that the enzymes act on fungal cell wall glycosidic linkages, likely having roles in cell wall remodelling. Both enzymes utilize an inverting mechanism but differ in specificity and product profiles indicating exo- and endo-like activity for AnGH71B and AnGH71C, respectively. We present the first structure of a GH71 protein, AnGH71C, including structures with carbohydrate ligands. These structures revealed a conserved acidic dyad (DxxE), found to be crucial for activity, and active site water coordination consistent with a classical inverting GH mechanism. This work provides new insights into GH71, highlighting its functional diversity and the enzymes roles in fungal physiology.