Biochem. J. (2006) 399
(241–247) (Printed in Great Britain)
Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a b-N-acetylglucosaminidase and not a hyaluronidase
William L. SHELDON*†, Matthew S. MACAULEY‡, Edward J. TAYLOR§, Charlotte E. ROBINSON*, Simon J. CHARNOCK*1, Gideon J. DAVIES§, David J. VOCADLO‡ and Gary W. BLACK*2
*Biomolecular and Biomedical Research Centre, School of Applied Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, U.K., †School of Health, Natural and Social Sciences, University of Sunderland, Sunderland SR1 3SD, U.K., ‡Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6, and §York Structural Biology Laboratory, Department of Chemistry, University of York, York YO31 5YW, U.K.
Key words: b-N-acetylglucosaminidase (GlcNAcase), mammalian glycoproteins, 1,2-dideoxy-2´-methyl-a-D-glucopyranoso-[2,1-d]-D2´-thiazoline (NAG-thiazoline), O-glycoprotein 2-acetamido-2-deoxy-b-D-glucopyranosidase (O-GlcNAcase), O-glycoprotein 2-acetamido-2-deoxy-b-D-glucopyranoside (O-GlcNAc), Spyl600, substrate-assisted catalysis.
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 b-O-linked N-acetylglucosamine from mammalian glycoproteins – an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-b-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-a-D-glucopyranoso-[2,1-d]-D2´-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 b-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.
Abbreviations used: GlcNAc, N-acetylglucosamine (2-acetamido-2-deoxy-D-glucopyranose); GlcNAcase, b-N-acetylglucosaminidase; MGEA5, meningioma expressed antigen 5; MU, 4-methylumbelliferyl; MU-GlcNAc, Mu-2-acetamido-2-deoxy-b-D-glucopyranoside; MU-GlcNAcF1, MU-2-deoxy-2-fluoroacetamido-b-D-glucopyranoside; MU-GlcNAcF2, MU-2-deoxy-2-difluoroacetamido-b-D-glucopyranoside; MU-GlcNAcF3, MU-2-deoxy-2-trifluoroacetamido-b-D-glucopyranoside; NAG-thiazoline, 1,2-dideoxy-2´-methyl-a-D-glucopyranoso-[2,1-d]-D2´-thiazoline; O-GlcNAc, O-glycoprotein 2-acetamido-2-deoxy-b-D-glucopyranoside; O-GlcNAcase, O-glycoprotein 2-acetamido-2-deoxy-b-D-glucopyranosidase; ORF, open reading frame.
1Present address: Megazyme International Ireland Limited, Bray, County Wicklow, Republic of Ireland.
2To whom correspondence should be addressed (email gary.black@northumbria.ac.uk).
Received 23 February 2006/26 June 2006; accepted 6 July 2006
Published as BJ Immediate Publication 6 July 2006, doi:10.1042/BJ20060307
The Biochemical Society, London ©2006
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