Deciphering Ligand Specificity of a Clostridium thermocellum Family 35 Carbohydrate Binding Module (CtCBM35) for Gluco- and Galacto- Substituted Mannans and Its Calcium Induced Stability

Abstract

This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (K a = 14.3×104 M−1), carob galactomannan (K a = 12.4×104 M−1) and negligible association (K a = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (K a = 41×104 M−1) and carob galactomannan (K a = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.

Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

“…Recently, we reported a CBM35 -CtCBM35 (i.e. Cthe 0032) from Clostridium thermocellum ATCC 27405 with similar type of ligand specificity (Ghosh et al 2013). However, the binding affinities of CtCBM35 (Cthe 2811) are apparently much higher than that of CtCBM35 (Cthe 0032).…”

“…Ligand specificity by CBM35 is probably due to the conserved hydrophobic aromatic residues that play a major role in polysaccharide binding (Tunnicliffe et al 2005). Ligand binding alters the microenvironment of tryptophan due to conformational changes in the CtCBM35 (Cthe 0032) (Ghosh et al 2013). The indole ring of tryptophan has intrinsic fluorescence properties with higher quantum yield and displays fluorescence emission at 320 nm to 400 nm (Royer 2006).…”

“…This means, both polysaccharides have single binding site for CtCBM35. Since CtCBM35 displayed significant affinities for mannose-derived polysaccharide in combination of galactose and glucose in their side and main chains, from the derived affinity constants the Gibb's free energy of binding were calculated using the equation as described earlier by Ghosh et al (2013). The free energy binding of CtCBM35 with carob galactomannan was −25.0 kJ/mol and −27.0 kJ/mol with konjac glucomannan (Table 1).…”

“…Multiple sequence alignment was carried out with appended CBM35 families GH5 and GH26 β-(1,4)-mannanases from Podospora anserine (PDB ID: 3ZM8; Couturier et al 2013), CBM35 from the Cellvibrio japonicus β-1,4-mannanase Man5C (PDB ID: 2BGO; Tunnicliffe et al 2005) and CBM35 (Cthe 0032) from Clostridium thermocellum ATCC 27405 (Ghosh et al 2013). Protein sequences were retrieved from PDB or CAZy databases, saved in FASTA format.…”

“…7c,d). The association constant (K a ) of CtCBM35 with carob galactomannan and konjac glucomannan were derived from modified Stern Volmer's equation (Ghosh et al 2013). From relative fluorescence intensities, the values of K a with carob galactomannan was 1.44 × 10 5 M −1 and with konjac glucomannan 2.40 × 10 5 M −1 (Table 1).…”