| Abstract |
Peptidoglycan is an important component of the bacterial cell wall.
Specifically, it a macromolecule comprised of long chains of alternating
saccharide units of N-acetylglucosamine and Ν-acetylmuramic acid linked by
β-1,4-N-glycoside bonds. 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. Deacetylases
of N-acetylglucosamine belong to the family 4 of carbohydrate esterases
(CE4), which includes N-acetylglucosamine peptidoglycan deacetylases,
rhizobial NodB chitooligosaccharide deacetylases, chitin deacetylases, acetylxylan esterases, and xylanases A, C, D and Ε 3. Members of this family are
composed from the highly conserved homology domain NodB, a well
conserved triad of metal binding amino acid residues (Asp, His, His), a wellconserved catalytic residue of Asp and a well conserved hydroxyl-Pro (2-Hyp)
in the Nodb domain. The genome of B. cereus has 6 peptidoglycan
deacetylases, three of them have already been studied (BC1960, BC3618,
BC1974). This study focusses on peptidoglycan deacetylase ΒC1960.
Specifically, a new point mutation of residue Arg169 to Ala was conducted, to
collect more data about the hydroxylation of Ca atom of Pro171 mechanism.
From the initial 13 clones, the 13th was chosen for further analysis. First of all,
the expression conditions were examined and determined. Then the protein was
overexpressed in BL12DE3 bacterial cells and purified using His-tag affinity
chromatography (Ni-NTA agarose). Subsequently, circular dichroism
experiments were held to collect structural data for the isolated protein.
According to these experiments the protein is characterized by a great thermostability as it remains in a folded state even in high. Also, it was shown that the
fusion protein is a typical a-helical protein as the required minimum and
maxima are visible. These results were verified by conducting SVD analysis.
The crystallization screens tested gave positive results, while at the same time
co-crystallization and crystal soaking experiments using three different ligands
were conducted.
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Δομική και βιοχημική μελέτη του ενεργού κέντρου της απακετυλάσης της πεπτιδογλυκάνης BC1960
