| Abstract |
Peptidoglycan is a macromolecule comprised of long chains of alternating saccharide units of GlcNAc-MurNAc, linked by β-1,4-N-glycoside bonds and it is an essential component of the bacterial cell wall. It can be subjected to modifications of its structure, such as N-deacetylation by the deacetylases of N-acetylglucosamine, which affects bacterial recognition by the host’s immunity system cells and also confers resistance to lysozyme. GlucNAc deacetylases belong to the family 4 of carbohydrate esterases (CE4), which includes N-acetylglucosamine peptidoglycan deacetylases (EC 3.1.1.-), rhizobial NodB chitooligosaccharide deacetylases (EC 3.5.1.-), chitin deacetylases (EC 3.5.1.41), acetyl-xylan esterases (EC 3.1.1.72), and xylanases A, C, D and Ε (EC 3.2.1.8). Members of this family are composed apart from the highy conserved homology domain NodB, from a well conserved triad of metal binding aminoacid residues (Asp, His, His), a well-conserved catalytic residue of Asp and a well conserved hydroxyl-Pro (2-Hyp) in the Nodb domain. The currently accepted catalysis mechanism of these metal-dependant enzymes depends on a acid-basis reaction mechanism. Genomes of Bacillus cereus ATCC 14579 as well of its close relative Bacillus anthracis (90% homology), contain 11 genes which encode for polysaccharide deacetylases, 6 of which encode for putative N-acetylglucosamine deacetylases which are highly homologous. Among these 6 GlucNAc deacetylases, BC2929 of Bacillus cereus can be found, which is the subject of this master thesis, and which shares 94% homology with BA2944 of Bacillus anthracis and also carries the conserved residues aforementioned but has Ser in the place of Pro in the active site. BC2929 (without signal peptide) was studied using diverse physicochemical methods with the aim of examining its properties and especially the formation of oligomers and aggregates. BC2929 was overexpressed in BL21 DE3 cells (with the presence of chaperones) as well as in JM109 cells (without chaperones) and in both cases a relatively medium level of solubility was observed and the protein was significantly unstable. The crystallization screens tested did not give any results. After gel filtration was performed, the presence of a dimer population showing deacetylase activity was observed. In addition, during protein concentration, aggregates were formed and were observed by TEM microscopy. The truncation of C-terminus of BC2929 seems to have reduced even more protein solubility and stability and to have affected aggregates’ formation. On the other hand, truncation of N-terminus affects polymerization. Its homologue ΒΑ2944 is found to be secreted to the cell surface, acting on neutral polysaccharide, while itself BC2929 is also predicted by a programme to be periplasmic. It is
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therefore suspected that it may be implicated in biofilm formation of Bacillus cereus. Results showed that BC2929 is an unstable protein that passes through different stages of oligomerism, while structural analysis and further experiments are necessary to clarify its function.
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Δομικός και βιοχημικός χαρακτηρισμός της απακετυλάσης πολυσακχαριτών BC2929, του Bacillus cereus
