Functional Characterization and Structural Basis of the beta-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris

beta-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membrane-bound beta-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/mu g, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a K-m value of 84.28 mu M at pH 7.0 and synthesized beta-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDP-glucose potentially bound to at least 11 residues via hydrogen bonds, pi-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of beta-1,3-glucan synthases in mushrooms.

Automated summary

In this study, the gene CMGLS encoding the membrane-bound beta-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned and analyzed. The three-dimensional structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time. These findings provide insights into the glucan synthesis process and catalytic mechanism of beta-1,3-glucan synthases in mushrooms.