Small-angle X-ray scattering based structure, modeling and molecular dynamics analyses of a family 5 glycoside hydrolase first endo-mannanase named as RfGH5_7 from Ruminococcus flavefaciens.

Endo-ß-1,4-mannanase named as RfGH5_7 from Ruminococcus flavefaciens cloned, expressed and purified earlier was structurally characterized in present study. The RaptorX modeled structure of RfGH5_7 showed a (ß/α)8 Triose-phosphate Isomerase (TIM) barrel fold. The Ramachandran plot assessment of RfGH5_7 showed that all amino acids fall in allowed region except one, Asn22 in the disallowed region. The superposition of RfGH5_7 modeled structure with its nearest homologues revealed that Glu154 acts as proton donor while Glu249 acts as nucleophile. Secondary structure of RfGH5_7 through Circular Dichroism (CD) analysis revealed 33.5% α-helices, 17% ß-strands and 49.5% random coils. Molecular Dynamic (MD) simulation showed Root Mean Square Deviation (RMSD), 0.67 nm and radius of gyration (Rg) between 1.9 nm and 1.85 nm. The binding interaction of mannotetraose on the surface of RfGH5_7 structure displayed polar interactions with His219, Tyr221, Trp278, Ser279 and Gly282 residues. Small-angle X-ray scattering (SAXS) analysis displayed the intact and monodispersed nature of the enzyme RfGH5_7. The radius of gyration (Rg) by Guinier analysis for globular shape was found to be 2.29 ± 0.09 nm and for rod-shape it was 0.95 ± 0.02 nm. Kratky plot confirmed that RfGH5_7 structure is compact and folded in solution. The ab initio derived dummy model of RfGH5_7 displayed single domain structure of yellow humped fish like shape. The RfGH5_7 modeled structure was well fitted with ab initio derived model from SAXS data. Communicated by Ramaswamy H. Sarma.

Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase (RfGH5_7) from Ruminococcus flavefaciens FD-1 v3.

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.