Computational and SAXS-based structure insights of pectin acetyl esterase (CtPae12B) of family 12 carbohydrate esterase from Clostridium thermocellum ATCC 27405

Abstract

Pectin is a complex form of polysaccharide and is composed of several structural components that require the concerted action of several pectinases for its complete degradation. In this study, in silico and solution structure of a pectin acetyl esterase (CtPae12B) of family 12 carbohydrate esterase (CE12) from Clostridium thermocellum was determined. The CtPae12B modelled structure, showed a new α/β hydrolase fold, similar to the fold found in the crystal structures of its nearest homologues from CE12 family, which differed from α/β hydrolase fold found in glycoside hydrolases. In the active site of CtPae12B, two loops (loop1 and loop6) play an important role in the formation of a catalytic triad Ser15-Asp187-His190, where Ser15 acts as a nucleophile. The structural stability of CtPae12B and its catalytic site was detected by performing molecular dynamic (MD) simulation which showed stable and compact conformation of the structure. Molecular docking method was employed to analyse the conformations of various suitable ligands docked at the active site of CtPae12B. The stability and structural specificity of the catalytic residues with the ligand, 4-nitrophenyl acetate (4-NPA) was confirmed by MD simulation of CtPae12B-4NPA docked complex. Moreover, it was found that the nucleophile Ser15, forms hydrophobic interaction with 4-NPA in the active site to complete covalent catalysis. Small angle X-ray scattering analysis of CtPae12B at 3 mg/mL displayed elongated, compact and monodispersed nature in solution. The ab initio derived dummy model showed that CtPae12B exists as a homotrimer at 3 mg/mL which was also confirmed by dynamic light scattering.

Communicated by Ramaswamy H. Sarma

Keywords:

• Clostridium thermocellum
• pectin acetyl esterase
• catalytic triad
• MD simulation
• SAXS

Acknowledgements

The research work was supported by Trilateral project with Indian Institute of Technology Bombay, Mumbai, India and All India Institute of Medical Sciences (AIIMS, New Delhi, India) under DBT-Twining project scheme with grant (No. BT/PR24786/NER/95/853/2017) from Department of Biotechnology, Ministry of Science and Technology, New Delhi, Government of India to AG. The authors are thankful to Dr. Ravishankar Ramachandran, Principal Scientist and his team member Md Afsar, CSIR-Central Drug Research Institute, Lucknow, India for providing the SAXS facility and Data collection. The authors are also thankful to IIT Guwahati for Param-Ishan Supercomputing facility and Centre for Environment, IIT Guwahati, for providing the DLS facility.

Disclosure statement

No potential conflict of interest was reported by the authors.