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

Research output: Contribution to journalArticlepeer-review

Abstract

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. Spy 1600, 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 Spy 1600 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]-Delta(2') -thiazoline as a competitive inhibitor reveals that Spy 1600 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.

Original languageEnglish
Pages (from-to)241-247
Number of pages7
JournalBiochemical journal
Volume399
DOIs
Publication statusPublished - 15 Oct 2006

Keywords

  • beta-N-acetylglucosaminidase (GlcNAcase)
  • mammalian glycoproteins
  • 1,2-dideoxy-2 '-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta 2 '-thiazoline (NAG-thiazoline)
  • O-glycoprotein 2-acetamido-2-deoxy-beta-D-glucopyranosidase (O-GIcNAcase)
  • O-glycoprotein 2-acetamido-2-deoxy-beta-D-glucopyranoside (O-GlcNAc)
  • Spy1600
  • substrate-assisted catalysis
  • O-GLCNACASE
  • CLOSTRIDIUM-PERFRINGENS
  • MECHANISM
  • GLUCOSAMINIDASE
  • SUBSTRATE
  • IDENTIFICATION
  • PARTICIPATION
  • PURIFICATION
  • RESIDUES
  • BINDING

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