Year: 2022
Type: dissertation
Abstract: The human gut contains a community of commensal bacteria, named gut microbiota, which are essential to breakdown the dietary polysaccharides indigestible by humans, by producing carbohydrate-active enzymes (CAZymes). The Bacteroidetes phylum possess polysaccharide utilization loci (PUL) that code for CAZymes, along with other proteins responsible for transport and uptake of dietary polysaccharides and host-derived glycans. The CAZymes are usually associated with non-catalytic ancillary modules, nominated carbohydrate-binding modules (CBMs), important for targeting carbohydrates. Multiple CAZymes and CBMs are still not well characterized and require further structural and functional studies. The main goal of the work reported in this thesis was to structurally characterize three putative family 32 CBMs identified in the genome of Bacteroides caccae, a member of the gut microbiota. These putative CBM32s are the non-catalytic ancillary modules found in glycoside hydrolases family 31 (BC03580) and in peptidases from the M60-like family (BC16100-C and BC07535-C). A preliminary three-dimensional (3D) structure of BC03580 (PUL 22) was solved by X-ray crystallography at a resolution of 2.97Å, revealing multiple regions of disordered electron density. In addition, the 3D structures of BC16100-C from PUL 53 and BC16100-C bound to GalNAc were obtained with a resolution of 1.67Å and 1.6Å, respectively. A prediction of the 3D structure of BC07535-C (PUL 35) was also modelled using the machine-learning approach of AlphaFold2. The comparison with a characterized CBM32, BT3015-C, revealed the conservation of a -sandwich fold in the three putative CBM32s and a lack of conservation of some amino acids belonging to the putative binding site in BC16100-C and BC07535-C. This could result in different modes of binding to their respective carbohydrates ligands, Tn and core 1 and core 2 O-glycans. These results pave the way to an increased knowledge on the function of these important modules and their influence in the action of the enzyme partners.
Author: Candeias, Alícia Isabel Martins
Institution: Universidade Nova de Lisboa
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7030316714