Functional Characterization And Structural Basis Of The β-1,3-Glucan Synthase CMGLS From Cordyceps Militaris

β-1,3-glucan synthases play a crucial role in fungal glucan synthesis,cell wall assembly,and mycelial growth.However,multi-transmembrane domains（over 14TMHs）and large molecular weight（over 100 k Da）result in extreme difficulty to obtainβ-1,3-glucan synthases with high purity for analyzing their crystal structures,which also hinder the understanding of detailed structures,catalytic properties and mechanisms.Therefore,in the present study,a C.militarisβ-1,3-glucan synthase gene CMGLS was cloned,re-annotated,and well bioinformatics analyzed.Subsequently,β-1,3-glucan synthase CMGLS was partially purified by product entrapment method,and its central hydrophilic cytoplasmic domain（r CMGLS-CHCD）was heterologously expressed and purified to systematically investigate their enzymatic properties.Finally,Alphafold 2 was applied to describe 3D structure of CMGLS for the first time,and molecular docking was used to elucidate its binding or catalytic sites with substrate UDP-glucose.These findings would provide a basis to reveal the synthesis process ofβ-1,3-glucan and their reasonable catalytic mechanism ofβ-1,3-glucan synthases in mushrooms.The main conclusions are as follows:（1）β-1,3-glucan synthase gene CMGLS in C.militaris was cloned,identified and structurally analyzed.Its full-length g DNA and c DNA sequences were 5949 bp and5841 bp,respectively.CMGLSp was composed of 1946 amino acids with a molecular mass of 221.70 k Da and no signal peptide.CMGLSp was a hydrophobic membrane protein belonging to GT48 family containing 15 transmembrane helices and three conserved domains including Glutenin＿hmw super family,FKS1 domain（pfam14288）and glucan synthase domain（pfam 02364）.CMGLS shared a high homology with other GLSs in Beauveria and Cordyceps.The central hydrophilic domains of these GLSs were also in high homology（750-1353aa）,while no putative UDP-glucose binding motif QXXRW or R/KXGG was observed in the central hydrophilic catalytic domain in CMGLSp.（2）β-1,3-glucan synthase CMGLS was obtained from cultivated mycelia of C.militaris using product entrapment method and its central hydrophilic cytoplasmic domain（r CMGLS-CHCD）was heterologously expressed and purified to systematically investigate their catalytic properties.The results showed that partially purifiedβ-1,3-glucan synthase CMGLS had a yield of 4.73%,the specific activity of87.72 pmo L/min/μg and a purification fold of 121.SDS-PAGE and LC-MS/MS results indicated that the partially purified CMGLS also contained a regulatory component Rho1.CMGLS had strict substrate specificity to UDP-glucose as the substrate while r CMGLS-CHCD showed an extremely low activity.The optimal p H and temperature of CMGLS were 7.0 and 37°C,respectively.CMGLS had the stable activity and the stability at temperature 20-42°C and p H 6.0-8.0 with the relative activity of approximate 80%.Under optimal reaction conditions,K_m,V_max and K_cat of CMGLS were 84.28±0.02μM,1.49±0.01μM/min and 1.42±0.01 min^-1,respectively.Metal ions Mg²⁺,K⁺,Fe²⁺,Cu²⁺,Zn²⁺,Co²⁺and Ca²⁺inhibited its activity by about 50%,while Fe³⁺at 1.0 m M enhanced the CMGLS activity by approximately 20%.（3）AlphaFold 2 and molecular docking were used to predict the 3D structure ofβ-1,3-glucan synthase CMGLS and its binding or catalytic sites with substrate UDP-glucose.HPLC and NMR results showed that CMGLS-synthesized products wereβ-1,3-glycosidic linked oligoglucoses or glucans with DPs 6-70.The 3D structure of C.militaris CMGLSp was constructed using Alpha Fold 2 for the first time webserver,containing the extrinsic,transmembrane,and periplasmic modules.TMH domains in CMGLS showed the neutral potential,while the negative and positive potentials were regionally distributed on the surfaces of extrinsic and periplasmic regions.With the help of molecular docking,the potential binding place for UDP-glucose in CMGLS was positioned in a pocket formed fromαhelixesα1,α2,α3 and the beta sheetβ1.UDP-glucose is docked to multiple amino acid residues in the CMGLS domain by hydrogen bonds,π-stacking,and salt bridges.Arg 1436 acted directly on moieties of glucose,phosphate and ribose ring on UDP-glucose,suggesting that it is a key residue playing an important role in binding substrates and/or catalyzing substrates.