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Identifier

000351285

Title

Κλωνοποίηση, έκφραση γονιδίων δύο νέων απακετυλασών χιτοολιγοσακχαριτών : απομόνωση, βιοχημικός χαρακτηρισμός

Alternative Title

Cloning and expression of two novel chitooligosaccharide deacetylases

Author

Δελή, Αλεξάνδρα

Thesis advisor

Μπουριώτης, Βασίσλης

Abstract

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain two LmbE protein family homologues. Only a few members of this family have been presently biochemically characterized including N-acetylglucosaminylphosphatidyl inositol, 1-D-myo-inosityl-2-acetamido-2-deoxy-α-D-glucopynanoside, diacetylchitobiose, and lipoglycopeptide antibiotic de-N-acetylases. All these enzymes share a common feature to de-N-acetylate the N-acetyl-D-glucosamine (GlcNAc) moiety of their substrates. The genes bc1534 and bc3461 from B. cereus ATCC14579 have been cloned and expressed in E. coli and the recombinant enzymes have been purified to homogeneity and further characterized. The enzymes exhibited different molecular masses, pH and temperature optima and a broad substrate specificity, de-N-acetylating GlcNAc, N-acetylchitooligomers (GlcNAc_2-4) as well as GlcNAc–1P, GlcNAc–6P, GalNAc, ManNAc, UDP-GlcNAc. However the enzymes were not active on radiolabeled glycol chitin, N-acetyl-D-glucosaminyl-(β-1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP), N-acetyl-D-GlcN-α1-6-D-myo-inositol-1-HPO₄-octadecyl (GlcNAc-I-P-C18). Kinetic analysis of BC1534, and BC3461 towards GlcNAc1-2 and docking experiments with selected substrates confirmed GlcNAc2 as the favoured one for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutagenesis analysis was carried out focusing on key residues. The catalytic efficiency of BC1534 variants was significantly decreased compared to the native enzyme. An alignment-based tree places both de-N-acetylases to different functional categories from other LmbE proteins.

Language

Greek

Subject

Basillus anthracis

Basillus cereus

Consensus sequence

GlcNac

LmbE

Κρυσταλλική δομή

Μεταβολικό μονοπάτι

Μεταλλαξογένηση

Ν,Ν'-διακετυλχιτοβιόζης