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

Abstract

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 Ka ~ 1.2·10(5) and 3.0·10(5) M(-1) with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with Ka 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 Ka.

The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome

“…1 D). The binding profile of BT0996-C contrasted with that obtained with a homologous β-mannan-specific CBM35 from Clostridium thermocellum (CtCBM35 Cthe_2811 ) ( Ghosh, et al, 2014 ), which showed the predicted binding to galactomannans ( Fig. 1 C).…”

A Recombinant β-Mannanase from Thermoanaerobacterium aotearoense SCUT27: Biochemical Characterization and Its Thermostability Improvement

“…aotearoense showed a typical (β/α) 8 barrel structure in GH5 and GH26 subfamilies. , The active sites of GH26 of Pa Man26A were identified in the groove, including Glu-300 and Glu-390 (Glu-328 and Glu-420 in Man25, respectively) positioned at the end of the barrel and several aromatic residues forming the subsites of the catalytic cleft . The CBM35 in Man25 showed a pair of six-stranded antiparallel β-sheets having a β-jelly-roll fold as many CBMs. , There were two metal ion binding sites conserved in most CBM35s; one is for carbohydrate recognition , and the other generally binds to Ca 2+ . Calcium ions could stabilize the protein structure by forming electrostatic interactions with amino acids, which may decrease the free hydrogen bonds or flexible loops at the N-terminus or C-terminus. , In this study, the calcium binding sites in Man25 were predicted as E32, E34, S53, G56, and D143 according to homology modeling.…”

“…18 The CBM35 in Man25 showed a pair of six-stranded antiparallel βsheets having a β-jelly-roll fold as many CBMs. 19,22 There were two metal ion binding sites conserved in most CBM35s; one is for carbohydrate recognition 3,18 and the other generally binds to Ca 2+ . 26 Calcium ions could stabilize the protein structure by forming electrostatic interactions with amino acids, which may decrease the free hydrogen bonds or flexible loops at the Nterminus or C-terminus.…”

“…β-Mannanase is available from many sources, including bacteria, fungi, actinomycetes, plants, and animals, − and can be classified into different glycoside hydrolase (GH) families (e.g., 5, 26, and 113 ,, ) according to the Carbohydrate-Active Enzymes database ( ). The families GH5, GH26, and GH113 belong to clan GH-A, and they share a similar protein folding pattern and catalytic mechanism. , Some mannanases possess an additional carbohydrate binding module (CBM), which could provide thermo-protection to the GH domain or alter the substrate specificity of the enzyme. , …”

“…15,16 Some mannanases possess an additional carbohydrate binding module (CBM), which could provide thermo-protection to the GH domain 17 or alter the substrate specificity of the enzyme. 18,19 For efficient bioconversion of lignocellulosic residues into biofuels and value-added products, thermostable enzymes are preferred during pretreatment, simultaneous saccharification, and fermentation. 20,21 Calcium ions often play a significant role in mannanase thermostabilization.…”