Ma Junwen, Qin Zhen, Zhou Peng, Wang Ruiming, Yan Qiaojuan, Jiang Zhengqiang, Yang Shaoqing
State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Sciences, Jinan 250353, China; Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, China.
School of Life Science Shanghai University, Shanghai 200237, China.
Enzyme Microb Technol. 2022 Jan;153:109948. doi: 10.1016/j.enzmictec.2021.109948. Epub 2021 Nov 15.
β-1,3-Glucan constitutes a prominent cell wall component being responsible for rigidity and strength of the cell wall structure in filamentous fungi. Glycoside hydrolase (GH) family 81 endo-β-1,3-glucanases which can cleave the long chain of β-1,3-glucans play a major role in fungal cell wall remodeling. Here, we reported the complex structures of a fungal GH family 81 endo-β-1,3-glucanase from Rhizomucor miehei (RmLam81A), revealing the triple-helical β-glucan recognition and hydrolysis patterns. In the crystals, three structured oligosaccharide ligands simultaneously interact with one enzyme molecular via seven glucose residues, and the spatial arrangement of ligands to RmLam81A was almost identical to that of β-1,3-glucan triple-helical structure. RmLam81A performed an inverting catalysis mechanism with Asp475 and Glu557 severing as the general acid and base catalyst, respectively. Furthermore, two hydrophobic patches involving Tyr93, Tyr106, Ile108, Phe619 and Tyr628 alongside the ligand-binding site possibly formed parts of the binding site. A ligand-binding motif, β31-β32, consisting of two key residues (Lys622 and Asp624), involved the recognition of a triple-helical β-glucan. Our results provided a structural basis for the unique β-1,3-glucan recognition pattern and catalytic mechanism of fungal GH family 81 endo-β-1,3-glucanases, which may be helpful in further understanding the diverse physiological functions of β-1,3-glucanases.
β-1,3-葡聚糖是丝状真菌细胞壁的主要成分，负责细胞壁结构的刚性和强度。糖苷水解酶（GH）家族81的内切β-1,3-葡聚糖酶能够切割β-1,3-葡聚糖的长链，在真菌细胞壁重塑中起主要作用。在此，我们报道了来自米根霉的真菌GH家族81内切β-1,3-葡聚糖酶（RmLam81A）的复杂结构，揭示了三螺旋β-葡聚糖的识别和水解模式。在晶体中，三个结构化的寡糖配体通过七个葡萄糖残基同时与一个酶分子相互作用，配体与RmLam81A的空间排列与β-1,3-葡聚糖三螺旋结构几乎相同。RmLam81A采用了一种转化催化机制，其中Asp475和Glu557分别作为广义酸和碱催化剂。此外，配体结合位点旁的两个疏水区域，涉及Tyr93、Tyr106、Ile108、Phe619和Tyr628，可能构成了结合位点的一部分。一个由两个关键残基（Lys622和Asp624）组成的配体结合基序β31-β32，参与了三螺旋β-葡聚糖的识别。我们的结果为真菌GH家族81内切β-1,3-葡聚糖酶独特的β-1,3-葡聚糖识别模式和催化机制提供了结构基础，这可能有助于进一步理解β-1,3-葡聚糖酶的多种生理功能。
