Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537391
Title: Biochemical and structural analyses of two hyaluronan lyases
Author: Elmabrouk, Zainab H.
Awarding Body: Northumbria University
Current Institution: Northumbria University
Date of Award: 2007
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Abstract:
Glycosaminoglycans are major components of the extracellular matrix, mostly in the form of proteoglycans. They are involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Hyaluronan and chondroitin sulphate comprise a biologically important subset of glycosaminoglycans. Hyaluronate lyases are glycosaminoglycan degrading enzymes that act as eliminases. They degrade hyaluronan the main polysaccharide component of the host connective tissues, thereby destroying the normal connective tissue structure and exposing the tissue cells to a variety of bacterial toxins. Two members of family 8 polysaccharide lyases were cloned, expressed in E. colt and purified to homogeneity using immobilised metal affinity and gel filtration chromatography methods. The first lyase was SC I C2.15 (82.9 kDa) from the soil bacterium Streptomyces coelicolor A3(2). Characterisation of the N-terminal hexahistidine tagged protein revealed that the enzyme displayed hyaluronate lyase activity, and it exhibited activity toward hyaluronan, chondroitin-4 and 6- sulphate with the highest activity toward HA. The enzyme displayed an optimum activity at pH 5.2 against hyaluronan and 4.8 against chondroitin-4 and 6-sulphate and an optimum temperature at 57 °C. The kinetic parameters of the enzyme against sodium hyaluronan, potassium hyaluronan, chondroitin-4-sulphate and chondroitin-6-sulphate were determined in the absence and presence of calcium. Additionally, HPAEC analysis showed that the enzyme has an exolytic mode of action. The enzyme was crystallised and its three-dimensional structure solved at a resolution of 2.7A. It was shown to be composed of two domains, an a-helical N-terminal catalytic domain and a f3- sheeted C-terminal domain. Through site directed mutagenesis, it was found that amino acid residues Tyr253, His244 and Asn194 are crucial for the enzyme, as substitution of these amino acids with alanine resulted in the loss of activity of the enzyme toward all substrates, demonstrating that these residues are pivotal in the catalysis by the Streptomyces coelicolor hyaluronate lyase. Y253A and N194A were co-crystallised with hyaluronan disaccharide, hyaluronan tetrasaccharide, chondroitin-4-sulphate disaccharide and L-ascorbic acid 6-hexadecanoate. The complexes were bound in the cleft located on the N-terminal domain of the enzyme in front of the interface between the N-and C- terminal domains revealing that the active site (substrate-binding site) is found in the cleft. The a and 13- domains of the Streptomyces coelicolor hyaluronate lyase were each cloned separately. Both subunits were expressed in insoluble form, and therefore both were refolded under denatured conditions. Characterisation of the N-terminal a-domain reveals the presence of small amounts of activity, representing approximately 10% of activity compared with the wild type enzyme, indicating that this domain is the functional domain of Streptomyces coelicolor hyaluronate lyase. Another ORF hylA of Streptococcus pyogenes SF370 that encodes hyaluronate lyase was cloned using a ligase independent cloning system and hyper-expressed in E. coll. Biochemical characterization of HylA showed that the enzyme is active only toward hyaluronan. It worked most efficiently at pH and temperature of 6 and 47 °C respectively, with a Km of 0.206 ± 0.01 mg nil-1 and a kat of 17.5 f 0.56 s-1. Moreover, the mode of action of the enzyme is endolytic as judged by analysis of digestion products by HPAEC. The enzyme was crystallised and sufficient quality diffraction data were derived enabling the determination of crystal parameters. Furthermore, four mutant proteins were generated via the mutagenesis strategy: Y327A, Y327F, N257A and H307A. These were expressed and purified in the same way as the native enzyme. The mutants Y327A, Y327F and N257A were completely inactive toward HA, while the mutant H307A showed very little activity against HA which was approximately 5%.
Supervisor: Black, Gary Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.537391  DOI: Not available
Keywords: C700 Molecular Biology, Biophysics and Biochemistry
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