Molecular and thermodynamic determinants of carbohydrate recognition by carbohydrate-binding modules and a bacterial pullulanase

dc.contributor.authorLammerts van Bueren, Alicia
dc.contributor.supervisorBoraston, Alisdair B. of Biochemistry and Microbiologyen_US of Philosophy Ph.D.en_US
dc.description.abstractProtein-carbohydrate interactions are pivotal to many biological processes, from plant cell wall degradation to host-pathogen interactions. Many of these processes require the deployment of carbohydrate-active enzymes in order to achieve their intended effects. One such class of enzymes, glycoside hydrolases, break down carbohydrate substrates by hydrolyzing the glycosidic bond within polysaccharides or between carbohydrates and non-carbohydrate moieties. The catalytic efficiency of glycoside hydrolases is often enhanced by carbohydrate-binding modules (CBMs) which are part of the modular structure of these enzymes. Understanding the carbohydrate binding function of these modules is often key to studying the catalytic properties of the enzyme. This thesis investigates the molecular determinants of carbohydrate recognition by CBMs that share similar amino acid sequences and overall three-dimensional structures and thus fall within the same CBM family. Specifically this research focused on two families; plant cell wall binding family 6 CBMs and the alpha-glucan binding family 41 CBMs. Through X-ray crystallography, isothermal titration calorimetry and other biochemical experiments, the structural and biophysical properties of CBMs were analyzed. Studying members of CBM family 6 allowed us to establish the overall picture of how similar CBMs interact with a diverse range of polysaccharide ligands. This was found to be due to changes in the topology of the binding site brought about by changes in amino acid side chains in very distinct regions of the binding pocket such that it adopted a three-dimensional shape that is complementary to the shape of the carbohydrate ligand. Members of CBM family 41 were shown to have nearly identical modes of starch recognition as found in starch-binding CBMs from other families. However family 41 CBMs are distinct as they are found mainly in pullulanases (starch debranching enzymes) and have developed binding pockets which are able to accommodate alpha-1,6-linkages, unlike other starch-binding CBM families. These are the first studies comparing multiple CBMs from within a given CBM family at the molecular level whose results allow us to examine the distinct modes of carbohydrate recognition within a CBM family. Analysis of the family 41 CBMs revealed that these CBMs are mainly found in pullulanases from pathogenic bacteria. Members from Streptococcal species were shown to specifically interact with glycogen stores within mouse lung tissue, leading us to investigate the role of alpha-glucan degradation by the pullulanase SpuA in the pathogenesis of Streptococcus pneumoniae. SpuA targets the alpha-1,6-branches in glycogen granules, forming alpha-1,4-glucan products of varying lengths. The overall three-dimensional structure of SpuA in complex with maltotetraose was determined by X-ray crystallography and showed that its active site architecture is optimal for interacting with branched substrates. Additionally, the N-terminal CBM41 module participates in binding substrate within the active site, a novel feature for CBMs. This is the first study of alpha-glucan degradation by a streptococcal virulence factor and aids in explaining why it is crucial for full virulence of the organism.en_US
dc.identifier.bibliographicCitationLammerts van Bueren A, Higgins M, Wang D, Burke RD and Boraston AB. (2007). Identification and structural basis of binding to host lung glycogen by streptococcal virulence factors. Nature Structural and Molecular Biology. 14(1):76-84.en_US
dc.identifier.bibliographicCitationLammerts van Bueren A and Boraston AB. (2007). The structural basis of alpha-glucan recognition by a family 41 carbohydrate-binding module from Thermotoga maritima. Journal of Molecular Biology. 365(3): 555-560.en_US
dc.identifier.bibliographicCitationLammerts van Bueren A, Morland C, Gilbert HJ, Boraston AB. (2005). Family 6 Carbohydrate Binding Modules Recognize the Non-reducing End of β-1,3-Linked Glucans by Presenting a Unique Ligand Binding Surface. Journal of Biological Chemistry. 280(1):530-537.en_US
dc.identifier.bibliographicCitationLammerts van Bueren A, Finn R, Ausio J, Boraston AB. (2004). Alpha-glucan recognition by a new family of carbohydrate-binding modules found primarily in bacterial pathogens. Biochemistry. 43(49):15633-15642.en_US
dc.identifier.bibliographicCitationLammerts van Bueren A, Boraston AB. (2004). Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites. Journal of Molecular Biology. 340(4):869-879.en_US
dc.rightsAvailable to the World Wide Weben_US
dc.subjectX-Ray crystallographyen_US
dc.subjectProtein-carbohydrate interactionen_US
dc.subjectcarbohydrate-binding moduleen_US
dc.subjectStreptococcus pneumoniaeen_US
dc.subjectglycoside hydrolaseen_US
dc.subject.lcshUVic Subject Index::Sciences and Engineering::Chemistry::Biochemistryen_US
dc.subject.lcshUVic Subject Index::Sciences and Engineering::Biology::Molecular biologyen_US
dc.titleMolecular and thermodynamic determinants of carbohydrate recognition by carbohydrate-binding modules and a bacterial pullulanaseen_US


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