Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a {beta}-N-acetylglucosaminidase and not a hyaluronidase

William L Sheldon , Matthew S Macauley , Edward J Taylor , Charlotte E Robinson , Simon J Charnock , Gideon J Davies , David J Vocadlo , Gary W Black (1)

1 School of Applied Sciences

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. Here 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-GlcNAcases within Glycoside Hydrolase family 84. Proton NMR studies, structure-reactivity studies for a series of fluorinated analogues, and analysis of NAG-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.

Mots clés: Life Sciences

William L Sheldon, Matthew S Macauley, Edward J Taylor, Charlotte E Robinson, Simon J Charnock, et al.. Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a {beta}-N-acetylglucosaminidase and not a hyaluronidase. Biochemical Journal, 2006, 399 (2), pp.241-247. ⟨10.1042/BJ20060307⟩. ⟨hal-00478542⟩