Nihira, T. | bioRxiv Science

bioRxiv2024

Beta-Glucanase superfamily identified by sequential, functional, and structural analyses

{beta}-1,2-Glucans are natural glucose polymers that play important physiological roles, including as symbiotic or pathogenic factors and in osmoregulation. Phylogenetically new glycoside hydrolase (GH) families have recently been identified from {beta}-1,2-glucanase (SGL) sequences from bacteria (GH144) and a fungus (GH162). In this study, we identified four phylogenetically new groups (Groups 1-4), and determined that these families, together with GH144, GH162, and GH189, a family of transglycosylase domains in cyclic {beta}-1,2-glucan synthases, form a superfamily. Biochemical analysis of six proteins in these groups revealed that the proteins in Groups 1-3 showed hydrolytic activity specific to {beta}-1,2-glucan. The kinetic parameters of the enzymes of Groups 1-3 were similar to GH144 and GH162 SGLs, indicating that these enzymes were SGLs. Optical rotation analysis revealed that the SGLs followed an anomer-inverting mechanism. Structural analysis and prediction of the proteins in Groups 1-4, GH144, GH162, and GH189 suggested that Groups 1-3 and GH144 had the same reaction mechanism. Nevertheless, Groups 1-3 were dispersed irregularly in the superfamily. Overall, we determined that Groups 1-3 were new GH families, GHxxx, GHyyy, and GHzzz, respectively, and proposed that this superfamily be called an SGL superfamily because of the phylogenetical, functional, and structural relationships within the superfamily. HighlightsWide variety of glycoside hydrolases is far beyond our understanding. Functional and structural analysis identified three new glycoside hydrolase families. Molecular evolution with irregular changes in reaction mechanism was revealed.

biochemistry↗

bioRxiv2022

Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan in Aspergillus oryzae

Aspergillus fungi contain -1,3-glucan with a low proportion of -1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored -amylases are conserved in Aspergillus fungi. The GPI-anchored -amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall -1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall -1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall -1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial -1,3-glucan. Furthermore, rAoAgtA cleaved 3--maltotetraosylglucose with a structure similar to the predicted boundary structure between the -1,3-glucan main chain and a short spacer composed of -1,4-linked glucose residues in cell wall -1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the -1,4-glycosidic bonds of 3--maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall -1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight (MW) of cell wall -1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored -amylases can degrade the spacer -1,4-glycosidic linkages in cell wall -1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall -1,3-glucan in vivo.

microbiology↗