Structure and Functional Investigation of Ligand Binding by a Family 35 Carbohydrate Binding Module (CtCBM35) of β-Mannanase of Family 26 Glycoside Hydrolase from Clostridium thermocellum

---

A. Ghosh^1#, A. K. Verma^1#, S. Gautam², M. N. Gupta², and A. Goyal^1*

¹Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India; fax: 91 (361) 2690762; E-mail: arungoyl@iitg.ernet.in; amalarastar@gmail.com

²Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, 110016, Delhi, India

^# These authors contributed equally.

^* To whom correspondence should be addressed.

Received August 31, 2013; Revision received February 26, 2014

---

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants K_a ~ 1.2·10⁵ and 3.0·10⁵ M^–1 with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with K_a of 5.0·10^–5 M^–1. Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of K_a.

---

KEY WORDS: CtCBM35, homology modeling, docking, CD spectra, affinity electrophoresis, fluorescence spectra, unfolding transition

DOI: 10.1134/S0006297914070098

---