Mechanisms by which Glycoside Hydrolases Recognize Plant, Bacterial and Yeast Polysaccharides A thesis submitted for the degree of Doctor of Philosophy Newcastle University 2008-2012 Fiona Cuskin Institute for Cell and Molecular Biosciences 1 Acknowledgements I would like to thank my supervisor Harry Gilbert, for the opportunity to study a PhD within his lab. I would also like to thank Harry, for his invaluable guidance, support and patience throughout my studies. The many people past and present I have worked with in Harry’s lab have provided invaluable advice and friendship and would like to thank Carl Morland, Dave Bolam, Elisabeth Lowe, Arthur Rogowski, Adam Jackson, Alan Cartmell, Lauren Mckee, Joanna Norman, Yanping Zhu and Wade Abbott to name a few. This work would not have been possible if it was not for the collaboration and expertise of Dr Arnaud Basle for the X-ray crystallography and Dr Alexandra Solovyova for the Analytical Ultracentrifugation. Finally, I would like to thank my family and friends for the love and support over the last 4 years. 2 Abstract The deconstruction of complex carbohydrates by glycoside hydrolases requires extensive enzyme consortia in which specificity is often conferred by accessory modules and domains that are distinct from the active site. The diverse mechanisms of substrate recognition were explored in this thesis using selected yeast, bacterial and plant polysaccharides as example substrates. Carbohydrate binding modules (CBM) are non-catalytic modules that enhance the catalytic activity of their glycoside hydrolase counterparts through binding to polysaccharide. Normally CBMs are found attached to glycoside hydrolases that target insoluble recalcitrant substrates resulting in a moderate, 2-5 fold, potentiation in enzyme activity. A CBM, defined herein as CBMX40, is found at the C-terminal of a glycoside hydrolase family (GH) 32 enzyme, SacC, which displays exo-levanase activity. CBMX40 binds the non-reducing end of the levan chain targeting the disaccharide fructose- -fructose unit. Removal of CBMX40 results in a >100-fold decrease in catalytic activity against levan, compared to the full length native enzyme. The truncated SacC catalytic domain acts as a non-specific exo-β-fructosidase displaying similar activity on β2,1- (inulin) and β2,6-linked fructose polymers, both polysaccharides and oligosaccharides. When CBMX40 was fused to a non-related exo- β-fructosidase, BT 3082, it conferred exo-levanase specificity on the enzyme. Thus CBMX40 is not only able to enhance catalytic activity but is also able to confer catalytic specificity. This led to the hypothesis that the CBM and the active site of the enzyme bind to different terminal residues of branched fructans such as levan. This results in enhanced affinity through avidity effects leading to the potentiation of catalytic activity. The gut bacterium Bacteroides thetaiotaomicron contributes to the maintenance of a healthy human gut. B. thetaiotaomicron is able to acquire and utilise complex carbohydrates that are not attacked by the intestinal enzymes of the host. B. thetaiotaomicron dedicates a large proportion of its genome to glycan degradation with a large expansion of α-mannan degrading enzymes. The B. thetaiotaomicron genome encodes 23 GH92 α- mannanosidases and 10 GH76 α-mannanases. While GH92 has recently been characterised the activities displayed by GH76 relies on the characterization of a single enzyme in this family. B. thetaiotaomicron organises the genes required to sense, degrade, transport and utilise specific complex glycans into genetic clusters defined as Polysaccharide Utilisation Loci (PULs). Transcriptomics revealed that two PULs are up regulated in response to yeast mannan, PUL 36 and PUL 68. These PULs contain both GH76 enzymes along with GH92 enzymes and other CAZy annotated enzymes. Biochemical analysis of the GH76 enzymes found in the two PULs show they are α1, 6 mannanases capable of hydrolysing the α1, 6 mannan backbone of yeast mannan, with the putative periplasmic enzymes generating small oligosaccharides, while the surface mannanases releasing larger products. The three GH92 enzymes encoded by the two PULs have been shown to remove α1, 2 and α1, 3 linked mannose branches from yeast mannan polysaccharide. In addition PUL 68 also encodes a phosphatase that removes the phosphate from mannose-6-phosphate and glucose-6-phosphate but not from intact mannan. Therefore, this study describes the ability of B. thetaiotaomicron to target and degrade yeast α-mannans. The GH5 enzyme CtXyl5A from Clostridium thermocellum is an arabinoxylan specific xylanase that contains a GH5 catalytic module appended to several CBMs. The apo structure of the GH5 catalytic module appended to a family 6 CBM reveals a large pocket abutted to the -1 subsite of the active site. This pocket was thought to bind the arabinose decoration appended to the O3 of the xylan backbone. Here mutational and structural studies showed that the fulfilment of arabinose is this pocket is the key specificity determinant for the novel arabinoxylanase activity. Significantly the bound arabinose displayed a pyranose conformation, rather than a furanose structure which is the typical conformation adopted by arabinose side chains in arabinoxylans. This structural information suggests that CtXyl5A may be able to exploit side chains other than arabinofuranose residues as substrate specificity determinants. 3 Contents Acknowledgements .................................................................................................... 2 Abstract ...................................................................................................................... 3 Contents ..................................................................................................................... 4 List of Figures ............................................................................................................. 8 List of Tables ............................................................................................................ 10 Chapter 1: Introduction ............................................................................................. 11 1.1 Complex polysaccharides structures and functions ................................... 12 1.1.1 Fructan polysaccharides ....................................................................... 12 1.1.2 Mannan from Saccharomyces cerevisiae ............................................. 17 1.1.3 Major plant structural polysaccharides ................................................. 20 1.2 Glycoside hydrolase ....................................................................................... 23 1.3 Carbohydrate Binding Modules....................................................................... 27 1.4 Glycoside Hydrolases acting on Fructan Polysaccharides ............................. 30 1.4.1 Family GH68 enzymes ......................................................................... 30 1.4.2 GH32 enzymes ..................................................................................... 31 1.5 Glycoside Hydrolase enzymes acting on Mannan polysaccharides................ 33 1.5.1 α-Mannosidases ................................................................................... 33 1.5.1.1 Glycoside hydrolase family 38 ....................................................... 33 1.5.1.2 Glycoside Hydrolase family 47 ....................................................... 36 1.5.1.3 Glycoside Hydrolase 92 ................................................................. 39 1.5.2 α-Mannanases (Glycoside hydrolase family 76) ................................... 40 1.6 Xylanases ....................................................................................................... 42 1.7 Bacteroides thetaiotaomicron ......................................................................... 45 1.7.1 The glycophilic nature of B. thetaiotaomicron ....................................... 45 1.7.2 Polysaccharide utilisation by B. thetaiotaomicron ................................. 46 1.7.2.1 Carbohydrate sensing and gene regulation ................................... 50 1.8 Objectives ....................................................................................................... 53 Chapter 2: Materials and Methods ........................................................................... 54 2.1 Molecular Biology ........................................................................................... 54 2.1.1 Bacterial Strains and Plasmids ............................................................. 54 2.1.2 Media and Growth Conditions .............................................................. 54 4 2.1.3 Selective Media .................................................................................... 54 2.1.4 Storage of DNA and Bacteria ............................................................... 55 2.1.5 Sterilisation ........................................................................................... 57 2.1.6 Chemicals, enzymes and Media ........................................................... 57 2.1.7 Centrifugation ....................................................................................... 57 2.1.8 Chemically competent E. Coli ............................................................... 57 2.1.9 Transformation of E. coli ....................................................................... 58 2.1.10 Replication of DNA and rapid small scale plasmid preparation