The role of enzymes and binding modules in the degradation of eukaryotic, microbial and plant cell walls

• Main Author: Temple, Max
• Published: University of Newcastle upon Tyne 2016
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706342

The microbial enzymes that depolymerize complex carbohydrates are of industrial significance particularly in the biofuels and biorefinery sectors. In the human large bowel glycan utilization plays a critical role in defining the composition of the human gut microbial community (microbiota) which, in turn, has a significant impact on health. A central feature of these processes is the specificity of the enzymes and the non-catalytic carbohydrate binding modules (CBMs) that contribute to glycan degradation. This thesis describes research designed to understand the mechanisms by which CBMs and glycoside hydrolases contribute to glycan degradation and how this impacts on the structure of the microbiota. The first results chapter describes the biochemical properties and structural basis for the specificity displayed by two CBMs appended to the glucanase of a rumen bacterium. The sequence of the two CBMs are >75% identical and display essentially identical ligand specificities. Isothermal titration calorimetry revealed that the two proteins bound to a range of β1,4-glucans (cellulose) and, β1,3-β1,4-mixed linked glucans, displaying highest affinity for xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This chapter reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans. In the second chapter the enzymes that contribute to the degradation of α-mannan, a prominent component of yeast cell walls, were studied. These enzymes were derived from Bacteroides thetaiotaomicron, a member of the microbiota. The data showed that the GH76 endo-α1,6-mannanases presented on the bacterial surface displayed significantly less activity against small mannooligosaccharides compared to the equivalent periplasmic enzymes. All the endo-α1,6-mannanases were only active on the linear backbone of a-mannan, the enzymes were unable to accommodate any side chains. These decorations were partially removed by a poorly expressed and slow acting surface GH92 α-mannosidase. In contrast, in the periplasm a highly active GH38 α-mannosidase rapidly debranched the imported yeast mannan oligosaccharides. The manooligosaccharides generated by the GH76 enzymes were then depolymerized into mannose by a pair of periplasmic exo-acting α1,6-mannosidases that contained only two substrate binding subsites. The biochemical characterization of these enzymes led to the selfish hypothesis in which B. thetaiotaomicron maximises deconstruction of yeast mannan in the periplasm, ensuring that the mannose generated will not be available to other organisms in the microbiota. This hypothesis were verified by showing that B. thetaiotaomicron was unable to support the growth of other Bacteroides sp. (that were able to grow on mannose and, in the case of B. xylanisolvens, also on debranched α-mannan) on yeast α-mannan. In the final results chapter the mechanism by which B. thetaiotaomicron utilized β1,6-glucan, a component of the yeast wall, was analysed. Transcriptomic analysis identified a Polysaccharide Utilization Locus (PUL) that was transcribed in response to β1,6-glucan. The PUL encoded two enzymes and two surface glycan binding proteins (SGBPs), one of which was a SusD homologue. The two SGBPs displayed tight specificity for β1,6-glucan over other β-glucans, displaying a preference for ligands that contained >3 glucose units. The surface GH30 enzyme, BT3312, was shown to be an endo-β1,6-glucanase, while the periplasmic GH3 exo-acting β-glucosidase displayed a preference for β1,6-linkages. B. thetaiotaomicron accumulated β1,6-glucobiose, which was due to the low activity of the GH3 enzyme against the disaccharide and poor expression of the β-glucosidase. The crystal structure of BT3312 revealed a deep pocket that mirrored the U-shaped typology of β1,6-glucan, revealing the mechanism of substrate specificity. Finally the catalytic amino acids of both the GH30 and GH76 enzymes were identified by site-directed mutagenesis.