Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537305
Title: Characterisation of hyaluronate lyases from streptococcal species
Author: Lindsay, Anna-Marie
Awarding Body: Northumbria University
Current Institution: Northumbria University
Date of Award: 2008
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Previously cloned bacteriophage encoded HyIP 1 from the genome sequenced organism Streptococcus pyogenes SF370 was expressed in Escherichia coli and purified to homogeneity. The protein, previously assigned to glycoside hydrolase family 69 (GH69) was biochemically recharacterised as a polysaccharide lyase and reassigned to family 16 (PL16). The enzyme demonstrated a Km of 1.47 mg m1-1 and a kcat of 7.2 s-1. Biochemically the enzyme had an optimum pH of 6.5 and temperature of 37 °C. The enzyme required no additional divalent ions for catalysis. The enzyme demonstrated strict substrate specificity only degrading hyaluronate and with no activity against related substrates chondroitin 4 sulphate and chondroitin 6 sulphate. HPAEC indicated the HylP1 had an endo mechanism of cleavage producing a range of differently sized oligosaccharides with the smallest being a tetramer. Site directed mutagenesis revealed a role for residues D157 and Y169 with substitution of these residues with alanine resulted in a 88.5% and 91.9 % loss of activity respectively. The location of these residues within the solved structure of HylP1 falls within the triple stranded (3 helix formed by the trimerised protein. This region of the protein was cloned, expressed and characterised and demonstrated similar kinetics as the full length protein (Km of 0.53 mg m1-1 and keat of 11.1 s-1). The activity of the enzyme when compared to other hyaluronate lyases shows it to be relatively inefficient yet when compared to other bacteriophage encoded hyaluronate lyases, HylP 1 was very similar. The proposed role of these bacteriophage encoded hyaluronate lyases is one of degradation of the hyaluronate capsule surrounding the streptococcal cells to allow for penetration of the bacteriophage during infection. Using the sequence of HylP1 the recently completed genome of Streptococcus equi was searched using bioinformatics tool BLAST. This revealed the presence of a protein, SEQ2045, sharing 85 % identity with HylPl. The protein was cloned and expressed in E. colt and biochemically characterised as a hyaluronate lyase. The enzyme demonstrated a Km of 2.05 mg m1-1 and a kcat of 6.2 s-1 which when compared to those of Hy1P 1 is suggestive that the two enzymes are strongly related. The enzyme had an optimum pH of 6.5 and temperature of 37 °C and like Hy1P 1 demonstrated only activity against hyaluronate and had an endo mechanism of cleavage with the smallest product of digestion being a tetramer. Site directed mutagenesis of the same residues as in HylP1 again yielded reduced activity (91.3 % and 87.6 % respectively). Bioinformatic analysis of the genome of S. equi was performed by BLAST searching with the proposed gene sequences of S. equi flanking SEQ2045. This allowed for the production of a prophage map which shows distinct similarities to the prophage map of S. pyogenes suggesting both may be of the same origin. Purified SEQ2045 was used in western blot analysis with S. equi convalescent horse serum. A strong positive reaction demonstrated a possible role for SEQ2045 during an infection suggesting that the bacteria have acquired this enzyme as a potential virulence factor by horizontal gene transfer. This presents a useful opportunity for the study of both S. pyogenes and S. equi infection process and the role of bacteriophage by the use of S. equi as a model for S. pyogenes.
Supervisor: Black, Gary Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.537305  DOI: Not available
Keywords: B900 Others in Subjects allied to Medicine ; C100 Biology ; nces
Share: