Biochemical and structural characterization of a glucan synthase GFGLS2 from edible fungus Grifola frondosa to synthesize β-1, 3-glucan

Background Grifola frondosa is a Basidiomycete fungus belonging to the family of Grifolaceae and the order of Polyporales. beta-Glucans are the main polymers in G. frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. The membrane-integrated beta-1, 3-glucan synthase (GLS) is responsible for glucan synthesis, cell wall assembly, differentiation and growth of the edible fungi. However, the structural/catalytic characteristics and mechanisms of beta-1, 3-glucan synthases in G. frondosa are still unknown due to their extremely complex structures with multi-transmembranes and large molecular masses.Results Herein, a beta-1, 3-glucan synthase (GFGLS2) was purified and identified from the cultured mycelia with a specific activity of 60.01 pmol min(-1 )mu g(-1 ) for the first time. The GFGLS2 showed a strict specificity to UDP-glucose with a V-max value of 1.29 +/- 0.04 mu M min(-1 ) at pH 7.0 and synthesized beta-1, 3-glucan with a maximum degree of polymerization (DP) of 62. Sequence Similarity Network (SSN) analysis revealed that GFGLS2 has a close relationship with others in Ganoderma sinense, Trametes coccinea, Polyporus brumalis, and Trametes pubescens. With the assistance of 3D structure modelling by AlphaFold 2, molecular docking and molecular dynamics simulations, the central hydrophilic domain (Class III) in GFGLS2 was the main active sites through binding the substrate UDP-glucose to 11 amino acid residues via hydrogen bonds, pi-stacking and salt bridges.Conclusions The biochemical, 3D structural characterization and potential catalytic mechanism of a membrane-bound beta-1, 3-glucan synthase GFGLS2 from cultured mycelia of G. frondosa were well investigated and would provide a reasonable full picture of beta-1, 3-glucan synthesis in fungi.