Molecular dynamics-based structural insights of the first putative endoglucanase, PsGH5A of glycoside hydrolase family 5 from Pseudopedobacter saltans.

CONTEXT: The putative endoglucanase, PsGH5A, from Pseudopedobacter saltans of family GH5 contains a catalytic module, PsGH5 (ß/α)8-TIM barrel), at N-terminal followed by a family 6 carbohydrate-binding module (CBM6, ß-sandwich). Superposition of PsGH5A with PDB homologs revealed Glu220 and Glu318 as evolutionarily conserved and catalytic residues performing the hydrolysis through retaining-type mechanism, a canonical property of GH5 family. PsGH5A showed higher affinity for longer cellooligosaccharides, as long as cellodecaose with binding free energy (∆G) of - 13.72 kcal/mol upon the molecular docking, thereby indicating the endo-mode of hydrolysis. The radius of gyration, Rg (2.7 nm), and solvent accessible surface area, SASA (229.6 nm2), of PsGH5A-Cellotetraose complex were determined by MD simulation which was lower than that of PsGH5A (Rg, 2.8 nm, SASA, 267 nm2) demonstrating the compactness and affinity of PsGH5A with the cellulosic ligands. Cellulose compatibility of PsGH5A was further confirmed by MMPBSA and per-residue decomposition analysis, where notable ∆G of - 54.38 kcal/mol for PsGH5A-Cellotetraose complex was observed. Thus, PsGH5A could be potentially an efficient endoglucanase as it accommodated larger cellooligosaccharides at its active-site. PsGH5A is the first putative endoglucanase studied here from P. saltans which could be genome-mined for lignocellulosic biomass saccharification in the renewable energy sector. METHODS: The 3-D structure of PsGH5A generated by AlphaFold2, RaptorX, SwissModel, Phyre2 and Robetta tool; YASARA was used for energy minimization of built models. UCLA SAVES-v6 was used for quality assessment of models. Molecular Docking was performed using SWISS-DOCK server and Chimera software. Molecular Dynamics simulations and MMPBSA analysis of PsGH5A and PsGH5A-Cellotetraose complex were performed on GROMACS 2019.6.

Computational design and structure dynamics analysis of bifunctional chimera of endoxylanase from Clostridium thermocellum and xylosidase from Bacteroides ovatus.

Development of chimeric enzymes by protein engineering can more efficiently contribute toward biomass conversion for bioenergy generation. Therefore, prior to experimental validation, a computational approach by modeling and molecular dynamic simulation can assess the structural and functional behavior of chimeric enzymes. In this study, a bifunctional chimera, CtXyn11A-BoGH43A comprising an efficient endoxylanase (CtXyn11A) from Clostridium thermocellum and xylosidase (BoGH43A) from Bacteroides ovatus was computationally designed and its binding and stability analysis with xylooligosaccharides were performed. The modeled chimera showed ß-jellyroll fold for CtXyn11A and 5-bladed ß-propeller fold for BoGH43A module. Stereo-chemical properties analyzed by Ramachandran plot showed 98.8% residues in allowed region, validating the modeled chimera. The catalytic residues identified by multiple sequence alignment were Glu94 and Glu184 for CtXyn11A and Asp229 and Glu384 for BoGH43A modules. CtXyn11A followed retaining-type, whereas BoGH43A enforced inverting-type of reaction mechanism during xylan hydrolysis as revealed by superposition and GH11 and GH43 familial analyses. Molecular docking studies showed binding energy, (ΔG) - 4.54 and - 4.18 kcal/mol for CtXyn11A and BoGH43A modules of chimera, respectively, with xylobiose, while - 3.94 and - 3.82 kcal/mol for CtXyn11A and BoGH43A modules of chimera, respectively, with xylotriose. MD simulation of CtXyn11A-BoGH43A complexed with xylobiose and xylotriose till 100 ns displayed stability by RMSD, compactness by R g and conformational stability by SASA analyses. The lowered values of RMSF in active-site residues, Glu94, Glu184, Asp229, Asp335 and Glu384 confirmed the efficient binding of chimera with xylobiose and xylotriose. These results were in agreement with the earlier experimental studies on CtXyn11A releasing xylooligosaccharides from xylan and BoGH43A releasing d-xylose from xylooligosaccharides and xylobiose. The chimera showed stronger affinity in terms of total short-range interaction energy; - 190 and - 121 kJ/mol for with xylobiose and xylotriose, respectively. The bifunctional chimera, CtXyn11A-BoGH43A showed stability and integrity with xylobiose and xylotriose. The designed chimera can be constructed and applied for efficient biomass conversion.

Multifunctionality and mechanism of processivity of family GH5 endoglucanase, RfGH5_4 from Ruminococcus flavefaciens on lignocellulosic polymers.

Multifunctional endoglucanase, RfGH5_4 from Ruminococcus flavefaciens showed (ß/α)8-TIM barrel structure by homology modeling. Glu168 and Glu292 residues acted as general acid and base during catalysis. Circular Dichroism results, 40.83 % α-helices, 13.84 % ß-strands and 45 % random turns-coils for RfGH5_4 corroborated with predictions by PSIPRED and SOPMA. Molecular Dynamic simulation of RfGH5_4 for 100 ns showed RMSD, 0.71 nm while for RfGH5_4-Cellopentaose complex was 0.55 nm, confirming that the binding of cellulosic ligand stabilizes its structural fold. RfGH5_4 showed strong affinity towards cellulosic ligands having higher degree of polymerization such as cellohexaose (-11.70 kcal/mol) and cellodecaose (-12.64 kcal/mol). Interestingly, complex hemicellulosic ligands such as XLLG of xyloglucan also showed higher affinity (-13.2 kcal/mol) and accommodated at RfGH5_4 active-site. Its catalytic cleft was broad enough to accommodate and hydrolyse various cellulosic and hemicellulosic ligands like XLLG of xyloglucan setting the basis of multifunctionality of RfGH5_4. Loops L2, L3 and L4 having Trp58 formed barrier at active-site of RfGH5_4 were responsible for processivity. RfGH5_4 showed monodispersed state at 2.5 mg/mL and a rattle-toy shape by SAXS. Zeta potential, -16 mV of RfGH5_4 indicated its higher stability. Multifunctional RfGH5_4 endoglucanase could be beneficial for generation lignocellulosic bioethanol and in health, prebiotic and food sector.

Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses.

Gene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 °C. It was stable between pH 5.0-8.0, retaining 85% activity and between 5 °C-45 °C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley ß-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg-1 s-1) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.