Κλωνοποίηση και έκφραση των γονιδίων bc2929 και bc5204 του οργανισμού Bacillus cereus: απομόνωση και βιοχημικός χαρακτηρισμός των ανασυνδυασμένων ενζύμων
Peptidoglycan is the basic polysaccharide of the bacterial cell wall and it is responsible for the stability and viability of the bacterial cell. It is a polymer of a repeating disaccharide-peptide unit (GlcNAc-MurNAc-pp), where the pentapeptide chains attached to adjacent sugar molecules are cross-linked. Peptidoglycan is a dynamic molecule and plays a crucial role in many biological functions of bacterial cells (bacterial growth, division, autolysis) through modifications of its structure. Such a modification is the deacetylation of peptidoglycan via the specialized enzymes peptidoglycan deacetylases. Peptidoglycan deacetylases belong to carbohydrate esterase family 4 (CE4) and de-N-acetylate the N-acetyl-muramic acid and N-acetyl-glucosamine residues of the di-sugar repeats in bacterial peptidoglycan. Through sequence alignments of CE4 esterases it has become apparent that all these members contain a highly conserved catalytic core, termed the NodB homology domain. CE4 family includes (according to Henrissat classification) chitin deacetylases, acetyl-xylan esterases, xylanases, rhizobial NodB chitooligosaccaride deacetylases and peptidoglycan deacetylases. A large number of open reading frames encoding for potential polysaccharide deacetylases have been identified in genomes of Gram-positive bacteria. The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 11 polysaccharide deacetylase homologues. Six of these have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Three of these genes (bc1960, bc3618 and bc1974) have already been cloned and expressed in Escherichia coli and the recombinant enzymes have been purified to homogeneity and further characterized. In the present work two additional genes, namely bc2929 and bc5204, have been cloned and expressed in Escherichia coli and the recombinant enzymes have been purified to homogeneity and biochemicaly characterized. Both enzymes can deacetylate chitin substrates, N-acetylchitooligosaccharides and the synthetic muropeptide N-acetyl-D-glucosamine-(β-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Kinetic analysis toward GlcNAc4-6 showed that GlcNAc6 was the favourable substrate for both enzymes. These results provide insight into the substrate specificity of carbohydrate esterase family 4 (CE4) enzymes. Considering the current interest in B. anthracis as a potential bioweapon and the difficulties encountered working with such agents (class A agent, Centre for Disease Control), these studies may also set a basis for potential drug design applications targeting enzymes involved in biosynthesis/modification of B. anthracis peptidoglycan.