Dataset Information

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ?-1,3-glucan.

ABSTRACT: BH0236 from Bacillus halodurans is a multimodular ?-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ?-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ?-1,3-glucan laminarin with a dissociation constant (Kd ) of ?26 ?m and displayed higher affinity for insoluble ?-1,3-glucans with Kd values of ?2-10 ?m but lacked affinity for ?-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ?-sandwich fold, with the face of one ?-sheet possessing the ?-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ?-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ?-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ?-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.

SUBMITTER: Hettle A

PROVIDER: S-EPMC5641894 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of β-1,3-glucan.

Hettle Andrew A Fillo Alexander A Abe Kento K Massel Patricia P Pluvinage Benjamin B Langelaan David N DN Smith Steven P SP Boraston Alisdair B AB

The Journal of biological chemistry 20170821 41

BH0236 from Bacillus halodurans is a multimodular β-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of β-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble β-1,3-glucan laminarin with a dissociation constant (Kd ) of ∼26 μm and displayed hig ...[more]

PMID: 28827308

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Project description:BackgroundEnzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment. Supplementation of xylanase in the cellulase mixture enhances glucose release from pretreated lignocellulose. Filamentous fungi produce multiple xylanases in their cellulase system, and some of them have modular structures consisting of a catalytic domain and a family 1 carbohydrate-binding module (CBM1). However, the role of CBM1 in xylanase in the synergistic hydrolysis of lignocellulose has not been investigated in depth.ResultsThermostable endo-β-1,4-xylanase (Xyl10A) from Talaromyces cellulolyticus, which is recognized as one of the core enzymes in the fungal cellulase system, has a modular structure consisting of a glycoside hydrolase family 10 catalytic domain and CBM1 at the C-terminus separated by a linker region. Three recombinant Xyl10A variants, that is, intact Xyl10A (Xyl10Awt), CBM1-deleted Xyl10A (Xyl10AdC), and CBM1 and linker region-deleted Xyl10A (Xyl10AdLC), were constructed and overexpressed in T. cellulolyticus. Cellulose-binding ability of Xyl10A CBM1 was demonstrated using quartz crystal microbalance with dissipation monitoring. Xyl10AdC and Xyl10AdLC showed relatively high catalytic activities for soluble and insoluble xylan substrates, whereas Xyl10Awt was more effective in xylan hydrolysis of wet disc-mill treated rice straw (WDM-RS). The enzyme mixture of cellulase monocomponents and intact or mutant Xyl10A enhanced the hydrolysis of WDM-RS glucan, with the most efficient synergism found in the interactions with Xyl10Awt. The increased glucan hydrolysis yield exhibited a linear relationship with the xylan hydrolysis yield by each enzyme. This relationship revealed significant hydrolysis of WDM-RS glucan with lower supplementation of Xyl10Awt.ConclusionsOur results suggest that Xyl10A CBM1 has the following two roles in synergistic hydrolysis of lignocellulose by Xyl10A and cellulases: enhancement of lignocellulosic xylan hydrolysis by binding to cellulose, and the efficient removal of xylan obstacles that interrupt the cellulase activity (because of similar binding target of CBM1). The combination of CBM-containing cellulases and xylanases in a fugal cellulase system could contribute to reduction of the enzyme loading in the hydrolysis of pretreated lignocelluloses.

Dataset Information

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ?-1,3-glucan.

Publications

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of β-1,3-glucan.

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