n-acetylglucosamine-thiazoline and 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-glucopyranoside

Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called 'flesh eating disease') and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove beta-O-linked N-acetylglucosamine from mammalian glycoproteins--an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-beta-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure-reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside hydrolases are therefore comprised of both prokaryotic and eukaryotic beta-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside hydrolases are most likely incorrectly annotated as hyaluronidases.

Topics: Acetylglucosamine; Acetylglucosaminidase; Amino Acid Sequence; Animals; beta-N-Acetylhexosaminidases; Catalysis; Chlorocebus aethiops; COS Cells; Escherichia coli; Histone Acetyltransferases; Humans; Hyaluronoglucosaminidase; Hydrolysis; Hymecromone; Kinetics; Molecular Sequence Data; Multienzyme Complexes; Nuclear Magnetic Resonance, Biomolecular; Sequence Homology, Amino Acid; Streptococcus pyogenes; Structure-Activity Relationship; Substrate Specificity; Thiazoles