Structure elucidation of a multi-modular recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC 27405 and its substrate binding analysis.

Autor: Mandal A; Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India., Ahmed J; Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India., Singh S; Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India; Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India., Goyal A; Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India. Electronic address: arungoyl@iitg.ac.in.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Jul; Vol. 273 (Pt 2), pp. 133212. Date of Electronic Publication: 2024 Jun 17.
DOI: 10.1016/j.ijbiomac.2024.133212
Abstrakt: Cellulases from GH9 family show endo-, exo- or processive endocellulase activity, but the reason behind the variation is unclear. A GH9 recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus was structurally characterized for conformation, binding and dynamics assessment. Modeled AtGH9C-CBM3A-CBM3B depicted (α/α) 6 -barrel structure with Asp98, Asp101 and Glu489 acting as catalytic triad. CD results revealed 25.2 % α-helix, 18.4 % β-sheet and rest 56.4 % of random coils, corroborating with predictions from PSIPRED and SOPMA. MD simulation of AtGH9C-CBM3A-CBM3B bound cellotetraose showed structural stability and global compactness with lowered RMSD values (1.5 nm) as compared with only AtGH9C-CBM3A-CBM3B (1.8 nm) for 200 ns. Higher fluctuation in RMSF values in far-positioned CBM3B pointed to its redundancy in substrate binding. Docking studies showed maximum binding with cellotetraose (ΔG = -5.05 kcal/mol), with reduced affinity towards ligands with degree of polymerization (DP) lower (DP < 4) or higher than 4 (DP > 4). Processivity index displayed the enzyme to be processive with loop 3 (342-379 aa) possibly blocking the non-reducing end of cellulose chain, resulting in cellotetraose release. SAXS analysis of AtGH9C-CBM3A-CBM3B at 5 mg/mL displayed monodispersed state with fist-and-elbow shape in solution. Negative zeta potential of -24 mV at 5 mg/mL indicated stability and free from aggregation.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Arun Goyal reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Databáze: MEDLINE
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