背景:灰树花是一种担子菌真菌,属于灰树花科和Polyporales科。β-葡聚糖是G.frondosa中的主要聚合物,在生理学中起着至关重要的作用,代表着人类的健康益处。膜整合的β-1,3-葡聚糖合酶(GLS)负责葡聚糖的合成,细胞壁组件,食用菌的分化和生长。然而,由于其具有多跨膜和大分子量的极其复杂的结构,G.frondosa中β-1,3-葡聚糖合酶的结构/催化特性和机理仍然未知。
结果:这里,首次从培养的菌丝体中纯化并鉴定了β-1,3-葡聚糖合酶(GFGLS2),比活性为60.01pmolmin-1μg-1。GFGLS2对UDP-葡萄糖具有严格的特异性,在pH7.0时的Vmax值为1.29±0.04µMmin-1,并合成了最大聚合度(DP)为62的β-1,3-葡聚糖。序列相似性网络(SSN)分析显示,GFGLS2与其它灵芝有密切的关系,Trametescoccinea,猪苓,和毛竹。借助AlphaFold2的3D结构建模,分子对接和分子动力学模拟,GFGLS2中的中心亲水结构域(III类)是通过氢键将底物UDP-葡萄糖与11个氨基酸残基结合的主要活性位点,π-堆叠和盐桥。
结论:生化,对培养的G.fordosa菌丝体的膜结合β-1,3-葡聚糖合酶GFGLS2的3D结构表征和潜在的催化机理进行了很好的研究,将为真菌中的β-1,3-葡聚糖合成提供合理的全貌。
BACKGROUND: Grifola frondosa is a Basidiomycete fungus belonging to the family of Grifolaceae and the order of Polyporales. β-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 β-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 β-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 β-1, 3-glucan synthase (GFGLS2) was purified and identified from the cultured mycelia with a specific activity of 60.01 pmol min-1 μg-1 for the first time. The GFGLS2 showed a strict specificity to UDP-glucose with a Vmax value of 1.29 ± 0.04 µM min-1 at pH 7.0 and synthesized β-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, π-stacking and salt bridges.
CONCLUSIONS: The biochemical, 3D structural characterization and potential catalytic mechanism of a membrane-bound β-1, 3-glucan synthase GFGLS2 from cultured mycelia of G. frondosa were well investigated and would provide a reasonable full picture of β-1, 3-glucan synthesis in fungi.