Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a beta-N-acetylglucosaminidase and not a hyaluronidase.
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.
Sheldon
William L
WL
Biomolecular and Biomedical Research Centre, School of Applied Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK.
Macauley
Matthew S
MS
Taylor
Edward J
EJ
Robinson
Charlotte E
CE
Charnock
Simon J
SJ
Davies
Gideon J
GJ
Vocadlo
David J
DJ
Black
Gary W
GW
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D004926:Escherichia coli; D051548:Histone Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000378:metabolism*; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017386:Sequence Homology, Amino Acid; D013297:Streptococcus pyogenes / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases
D009097:Multienzyme Complexes; D013844:Thiazoles; D006923:Hymecromone; C043647:4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-glucopyranoside; D051548:Histone Acetyltransferases; D006821:Hyaluronoglucosaminidase; C110628:hexosaminidase C; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; C487818:N-acetylglucosamine thiazoline; D000117:Acetylglucosamine