Small angle X-ray scattering based structure, modeling and molecular dynamics analyses of family 43 glycoside hydrolase α-L-arabinofuranosidase from Clostridium thermocellum

Abstract

Enzymes that participate in the hydrolysis of complex carbohydrates display a modular architecture, although the significance of enzyme modularity to flexibility and catalytic efficacy is not fully understood. α-L-arabinofuranosidase from Clostridium thermocellum (CtAraf43) catalyzes the release of α-1,2-, α-1,3-, or α-1,5- linked L-arabinose from arabinose decorated polysaccharides. CtAraf43 comprises an N-terminal catalytic domain (CtAbf43A) connected with two family 6 carbohydrate-binding modules (CBMs), termed as CtCBM6A and CtCBM6B, through flexible linker peptides. Here, we modeled the structure of CtAraf43 revealing that the module, CtAbf43A displays a 5-fold β-propeller fold and the CBMs the typical jellyroll type β-sandwich folds. Ramachandran plot showed 98.5% residues in the favored region and 1.5% residues in the disallowed region. Molecular dynamics simulation analysis of CtAraf43 revealed significant flexibility that is more expressive in the C-terminal CtCBM6B module in terms of structure and orientation. Small angle X-ray scattering analysis of CtAraf43 revealed its elongated structure. CtAraf43 at 1.2 mg/mL demonstrated the monomeric nature and a multi-modular shaped molecular envelope in solution with a Dmax of 12 nm. However, at 4.7 mg/mL, CtAraf43 displayed a dimeric structure and elongated molecular envelope. Kratky plot analysis revealed the folded state of CtAraf43 with limited flexibility at both concentrations. The data revealed higher flexibility at the C-terminal of CtAraf43 suggesting a coordinated action of the N-terminal catalytic module CtAbf43A and the internal CtCBM6A.AbbreviationCBMsCarbohydrate Binding ModulesCtAraf43α-L-arabinofuranosidaseGHsGlycoside HydrolasesMDMolecular DynamicsRMSDRoot Mean Square DeviationRMSFRoot Mean Square FluctuationSAXSSmall angle X-ray scatteringCommunicated by Ramaswamy H. Sarma