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 (β/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 % β-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.