Analysis of a family 6 carbohydrate-binding module and three family 69 hyaluronidases

Smith, Nicola L. (2005) Analysis of a family 6 carbohydrate-binding module and three family 69 hyaluronidases. Doctoral thesis, Northumbria University.

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Abstract

To investigate the interactions between a family 6 carbohydrate-binding module (CBM) from Clostridium thermocellum Xynl 1A (CtCBM6) and its target ligands and to identify the location of the ligand binding site(s) through a mutagenesis strategy, the protein was expressed in Escherichia coli and purified to homogeneity. CtCBM6 was shown previously to interact with xylan (Fernandes et al., 1999) and, informed by the crystal structure, it was found that CtCBM6 was unusual, as it contained two potential ligand-binding clefts (Clefts A and B). Qualitative ligand specificity studies through affinity gel electrophoresis (AGE) demonstrated that CtCBM6 bound preferentially to xylans, interacts weakly with 13-glucan and some soluble substituted forms of cellulose. Quantitative analysis of ligand binding by isothermal titration calorimetry showed that CtCBM6 bound xylooligosaccharides from xylobiose to xylohexaose, with affinity increasing with chain length. The affinity of CtCBM6 for soluble xylan of varying degrees of substitution was judged to be similar. NMR spectroscopy (Dr M. Czjzek at CNRS, Marseille) indicated that xylohexaose interacts with the two solvent exposed aromatic amino acids (Tyr-34 and Trp-92) and a polar amino acid (namely Asn-120) in cleft A of CtCBM6. Site-directed mutagenesis revealed that hydrophobic stacking interactions and hydrogen bonds potentiate the binding of CtCBM6 to xylan. Surface aromatic residues Tyr-34 and Trp-92 of CtCBM6 are pivotal in the interaction between this module and its ligand, as substitution of these amino acids with alanine and methionine resulted in an 8-fold and 50-fold respective decrease in affinity of CtCBM6 for oat spelt xylan, as judged by quantitative AGE. Hydrogen-bonding interactions also made pivotal contributions to the overall binding in CtCBM6. Asn-120 was critical to ligand binding, as the mutant N120A showed —130-fold loss of binding affinity. This suggests that this residue directly participates in ligand binding via hydrogen bonds. Collectively, mutagenesis and NMR studies showed that cleft A can accommodate xylooligosaccharides and xylan, while cleft B was unable to interact with target ligands. Three hyaluronidases (Hy1P1, HylP2 and HylP3) of glycoside hydrolase family 69 (GH69) were cloned from the genome sequenced organism Streptococcus pyogenes SF370, expressed in E. coli and purified to homogeneity. Characterisation of the N-terminally tagged HylP1 (38.4 kDa), Hy1P2 (42.0 kDa) and Hy1133 (41.8 kDa) revealed activity against sodium hyaluronate with a KM for Hy1P1, HylP2 and Hy1P3 of 0.90, 2.07 and 4.35 ml mg 1, and a kcat of 1390.90, 742.01 and 1253.04 s-1, respectively. HylP1, HylP2 and Hy1P3 displayed an optimum pH of 6.5, 6.0 and 5.5, respectively, and an optimum activity at 37 °C. Moreover, PAGE analysis showed each enzyme was endo-cleaving. All three enzymes have been crystallised and sufficient quality diffraction data obtained for Hy1P1 and HylP3 (data collection and processing was performed by Dr Edward Taylor). The 3D structure of HylP1 has been solved at a resolution of 1.8 A and is composed of three monomeric strands that are intertwined to form a trimer.

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