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 (beta/alpha)8-TIM barrel structure by homology modeling. Glu168 and Glu292 residues acted as general acid and base during catalysis. Circular Di-chroism results, 40.83 % alpha-helices, 13.84 % beta-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.

Automated summary

In this study, the multifunctional endoglucanase RfGH5_4 from Ruminococcus flavefaciens was found to have a (beta/alpha)8-TIM barrel structure through homology modeling. Experimental analyses confirmed the predicted secondary structure of RfGH5_4 and molecular dynamic simulation revealed the stability of its structural fold when binding to a cellulosic ligand. RfGH5_4 showed strong affinity towards cellulosic and hemicellulosic ligands, suggesting its potential in various industrial applications. The study also highlighted the role of specific loops in the catalytic cleft of RfGH5_4 and its monodispersed state and stability.