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.
Abbreviation | ||
CBMs | = | Carbohydrate Binding Modules |
CtAraf43 | = | α-L-arabinofuranosidase |
GHs | = | Glycoside Hydrolases |
MD | = | Molecular Dynamics |
RMSD | = | Root Mean Square Deviation |
RMSF | = | Root Mean Square Fluctuation |
SAXS | = | Small angle X-ray scattering |
Communicated by Ramaswamy H. Sarma
Acknowledgements
The authors are thankful to ESRF, Grenoble France for providing the SAXS data collection facility. Department of Biotechnology, Ministry of Science and Technology for DBT-CREST Fellowship to support the research work and AG.
Author’s contribution
AG, SN, and CF designed the study. AG prepared the sample for SAXS analysis. SN did the SAXS data collection. KS performed the SAXS data analysis and modeling and molecular dynamics analysis of CtAraf43. AG, SN, and KS wrote the paper.
Disclosure statement
No potential conflict of interest was reported by the author(s).