Functional characterization of a mixed-linkage beta-glucan utilization locus from the human gut microbe Prevotella copri
department or school: Science, Faculty of
department or school: Chemistry, Department of
data provider: DSpace
degree campus: UBCV
creator: Golisch, Benedikt
issued: 2024-09-30T07:00:00Z
issued: 2023
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: 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 GH9…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/85478
note: Functional characterization of a mixed-linkage beta-glucan utilization locus from the human gut microbe Prevotella copri by Benedikt Golisch B.S., Humboldt Universität zu Berlin, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2023 © Benedikt Golisch, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Functional characterization of a mixed-linkage beta-glucan utilization locus from the human gut microbe Prevotella copri submitted by Benedikt Golisch in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Examining Committee: Dr. Harry Brumer, Professor, Dept. of Chemistry, Dept. of Biochemistry and Molecular Biology, Dept. of Botany, UBC Supervisor Dr. Reinhard Jetter, Professor, Dept. of Chemistry, Dept. of Botany, UBC Supervisory Committee Member Dr. David Perrin, Professor, Dept. of Chemistry, UBC Supervisory Committee Member Dr. Chris Orvig, Professor, Dept. of Chemistry, UBC Additional Examiner iii Abstract 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 ge-netic, 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 s…
has type: Thesis/Dissertation
date issued: 2023-11
is shown at: 10.14288/1.0435198
language: eng
degree discipline: Chemistry
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Brumer, Harry
type: Text
identifier URI: http://hdl.handle.net/2429/85478