title: Synthesis of β-(1→6)-linked N-acetyl-D-glucosamine oligosaccharide substrates and their hydrolysis by Dispersin B.
abstract: Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.
authors.affiliation: Research Group for Carbohydrates of Hungarian Academy of Sciences, PO Box 94, H-4010 Debrecen, Hungary.
authors.forename: Anikó
authors.initials: A
authors.lastname: Fekete
authors.forename: Anikó
authors.initials: A
authors.lastname: Borbás
authors.forename: Gyöngyi
authors.initials: G
authors.lastname: Gyémánt
authors.forename: Lili
authors.initials: L
authors.lastname: Kandra
authors.forename: Erika
authors.initials: E
authors.lastname: Fazekas
authors.forename: Narayanan
authors.initials: N
authors.lastname: Ramasubbu
authors.forename: Sándor
authors.initials: S
authors.lastname: Antus
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry; D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001673:Biodegradation, Environmental; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development; D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism* / Q000494:pharmacology; D006027:Glycosides / Q000138:chemical synthesis / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000378:metabolism; D058105:Polymerization; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D006027:Glycosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014443:Tyrosine; D014364:Tryptophan; D006026:Glycoside Hydrolases; C501330:dispersin B, Actinobacillus actinomycetemcomitans; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine