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Golisch, Benedikt

University of British Columbia

The human gut microbiota (HGM) is a complex microbial community that possesses a huge capacity for carbohydrate degradation in contrast to humans and is dominated by the phylum Bacteroidetes comprising Bacteroides and Prevotella. While Bacteroides species are associated with people consuming protein/fat-rich diets, members of Prevotella including Prevotella copri are highly abundant in people consuming plant-based diets. Here, I present a comprehensive genetic, biochemical, and structural-biological investigation of a recently identified gene cluster of P. copri upregulated in response to mixed-linkage β-glucan (MLG), a polysaccharide commonly found in cereals. The MLG utilization locus (MLGUL) contains an SGBP-A that interacted with MLG specifically and an SGBP-B that recognized 1,4-linked β-glucans promiscuously. The tertiary structure of SGBP-A revealed an aromatic binding platform and flexible loop that interact with cellopentaose or potential MLG-breakdown products. Similarly, a structural model suggested a binding platform for the SGBP-B. Furthermore, the MLGUL encodes a GH5_4, a highly specific, endo-acting MLGase, that produced oligosaccharides by preferential hydrolysis of β-1,3-linkages. The product analysis and tertiary structure revealed that β-1,4-linked glucose units in the negative subsites and occupation of the +2 subsite facilitate this cleavage. In addition, the MLGUL includes an exo-acting β-glucosidase from the GH3 family that degraded longer oligosaccharides into glucose and cellobiose. The latter will be further cleaved to glucose and glucose 1-phosphate by a predicted cellobiose phosphorylase, a member of GH94. Together, these enzymological and biochemical studies support a model for MLG degradation by P. copri in which SGBPs bind substrate at the cell surface to facilitate cleavage by the endo-MLGase GH5_4 and import by the TonB-dependent transporter. The generated oligosaccharides will be sequentially cleaved by a GH3 β-glucosidase and a GH94 cellobiose phosphorylase, in the periplasm and cytosol, respectively, generating glucose and glucose 1-phosphate as final products for the metabolism of P. copri. Thus, the work provides insights into the molecular mechanism of MLG utilization by P. copri and might indicate factors affecting the fitness of P. copri within the HGM. This knowledge might advance our understanding of diet-host-microbe interactions and may result in innovative therapeutic approaches for prevalent diseases linked to the human gut.

Text

2024-09-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85478

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/