Combined SAXS and computational approaches for structure determination and binding characteristics of Chimera (CtGH1-L1-CtGH5-F194A) generated by assembling β-glucosidase (CtGH1) and a mutant endoglucanase (CtGH5-F194A) from Clostridium thermocellum

Chimera (CtGH1-L1-CtGH5-F194A) developed by fusing 13-glucosidase (CtGH1) at N-terminal and endoglucanase (CtGH5-F194A) at C-terminal was structurally characterized. Its secondary structure analysis by CD showed 38% alpha-helix, 9.3% beta-sheets and 52.7% random coils corroborating with prediction. In-silico modeled structure of Chimera comprised two modules, CtGH1 and CtGH5-F194A displaying (alpha/beta)(8) fold. Ramachandran plot of Chimera showed 99.9% residues in allowed region. Binding interaction of Chimera with cello-oligosaccharides suggested active forms of CtGH1 and CtGH5-F194A and their involvement in catalysis. MD simulation of cellohexaose bound endoglucanase module of Chimera showed favourable flexibility in loops, LA with H-bond formation with Asn510 and in loop LC relocation of Tyr687 away from active site efficiently releasing the product after catalysis. Higher short range interaction energy of Chimera, -383 kJ/mot than the individual endoglucanase, 254 kJ/mol against cellohexaose suggested higher efficient catalysis by Chimera.beta-Glucosidase module of Chimera showed fluctuations in outer loops suggesting conformational changes that might be contributing to improved hydrolysis. SAXS analysis of Chimera displayed monodispersed state. Guinier analysis of Chimera showed globular shape (R-g = 3.15 +/- 0.10 nm). Kratky plot confirmed fully folded and flexible behaviour in solution. Gasbor modeled structure of Chimera displayed an elongated structure with two modules having shape similar to bean-bag contour. (C) 2020 Elsevier B.V. All rights reserved.