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Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs.

ABSTRACT: BACKGROUND:A vast number of organisms are known to produce structurally diversified cellulases capable of degrading cellulose, the most abundant biopolymer on earth. The generally accepted paradigm is that the carbohydrate-binding modules (CBMs) of cellulases are required for efficient saccharification of insoluble substrates. Based on sequence data, surprisingly more than 60% of the cellulases identified lack carbohydrate-binding modules or alternative protein structures linked to cellulases (dockerins). This finding poses the question about the role of the CBMs: why would most cellulases lack CBMs, if they are necessary for the efficient hydrolysis of cellulose? RESULTS:The advantage of CBMs, which increase the affinity of cellulases to substrates, was found to be diminished by reducing the amount of water in the hydrolytic system, which increases the probability of enzyme-substrate interaction. At low substrate concentration (1% w/w), CBMs were found to be more important in the catalytic performance of the cellobiohydrolases TrCel7A and TrCel6A of Trichoderma reesei as compared to that of the endoglucanases TrCel5A and TrCel7B. Increasing the substrate concentration while maintaining the enzyme-to-substrate ratio enhanced adsorption of TrCel7A, independent of the presence of the CBM. At 20% (w/w) substrate concentration, the hydrolytic performance of cellulases without CBMs caught up with that of cellulases with CBMs. This phenomenon was more noticeable on the lignin-containing pretreated wheat straw as compared to the cellulosic Avicel, presumably due to unproductive adsorption of enzymes to lignin. CONCLUSIONS:Here we propose that the water content in the natural environments of carbohydrate-degrading organisms might have led to the evolution of various substrate-binding structures. In addition, some well recognized problems of economical saccharification such as unproductive binding of cellulases, which reduces the hydrolysis rate and prevents recycling of enzymes, could be partially overcome by omitting CBMs. This finding could help solve bottlenecks of enzymatic hydrolysis of lignocelluloses and speed up commercialization of second generation bioethanol.

SUBMITTER: Varnai A

PROVIDER: S-EPMC3599012 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs.

Várnai Anikó A Siika-Aho Matti M Viikari Liisa L

Biotechnology for biofuels 20130226 1

<h4>Background</h4>A vast number of organisms are known to produce structurally diversified cellulases capable of degrading cellulose, the most abundant biopolymer on earth. The generally accepted paradigm is that the carbohydrate-binding modules (CBMs) of cellulases are required for efficient saccharification of insoluble substrates. Based on sequence data, surprisingly more than 60% of the cellulases identified lack carbohydrate-binding modules or alternative protein structures linked to cellu ...[more]

PMID: 23442543

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Project description:Ordered water molecules bound to protein surfaces, or in protein-ligand interfaces, are frequently observed by crystallography. The investigation of the impact of such conserved water molecules on protein stability and ligand affinity requires detailed structural, dynamic, and thermodynamic analyses. Several crystal structures of the legume lectin concanavalin A (Con A) bound to closely related carbohydrate ligands show the presence of a conserved water molecule that mediates ligand binding. Experimental thermodynamic and theoretical studies have examined the role of this conserved water in the complexation of Con A with a synthetic analog of the natural trisaccharide, in which a hydroxyethyl side chain replaces the hydroxyl group at the C-2 position in the central mannosyl residue. Molecular modeling earlier indicated (Clarke, C.; Woods, R. J.; Glushka, J.; Cooper, A.; Nutley, M. A.; Boons, G.-J. J. Am. Chem. Soc. 2001, 123, 12238-12247) that the hydroxyl group in this synthetic side chain could occupy a position equivalent to that of the conserved water, and thus might displace it. An interpretation of the experimental thermodynamic data, which was consistent with the displacement of the conserved water, was also presented. The current work reports the crystal structure of Con A with this synthetic ligand and shows that even though the position and interactions of the conserved water are distorted, this key water is not displaced by the hydroxyethyl moiety. This new structural data provides a firm basis for molecular dynamics simulations and thermodynamic integration calculations whose results indicate that differences in van der Waals contacts (insertion energy), rather than electrostatic interactions (charging energy) are fundamentally responsible for the lower affinity of the synthetic ligand. When combined with the new crystallographic data, this study provides a straightforward interpretation for the lower affinity of the synthetic analog; specifically, that it arises primarily from weaker interactions with the protein via the positionally perturbed conserved water. This interpretation is fully consistent with the experimental observations that the free energy of binding is enthalpy driven, that there is both less enthalpic gain and less entropic penalty for binding the synthetic ligand, relative to the natural trisaccharide, and that the entropic component does not arise from releasing an ordered water molecule from the protein surface to the bulk solvent.

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Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs.

Publications

Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs.

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