title: Characterisation of a novel homodimeric N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii.
abstract: An N-acetyl-beta-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-beta-D-glucosaminide, 4MU-N-acetyl-beta-D-galactosaminide, 4-MU-beta-D-N,N'-diacetylchitobioside, and 4-MU-beta-D-N,N',N''-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 degrees C. The Km for 4-MU-beta-D-N,N',N''-chitotrioside, 45 microM, was the lowest for all the substrates tested. Hg2+, Cu2+, Fe2+, and Zn2+ completely inhibited while Co2+, Mn2+, and Ni2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-beta-D-glucosaminidase activities.
authors.affiliation: Institute of Dental Research, Millennium Institute, Westmead Centre for Oral Health, Westmead, Australia. dharty@dental.wsahs.nsw.gov.au
authors.forename: Derek W S
authors.initials: DW
authors.lastname: Harty
authors.forename: Yingjian
authors.initials: Y
authors.lastname: Chen
authors.forename: Christine L
authors.initials: CL
authors.lastname: Simpson
authors.forename: Tracey
authors.initials: T
authors.lastname: Berg
authors.forename: Simon L
authors.initials: SL
authors.lastname: Cook
authors.forename: John A
authors.initials: JA
authors.lastname: Mayo
authors.forename: Neil
authors.initials: N
authors.lastname: Hunter
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Jacques
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000201:enzymology*
chemical_list: D000118:Acetylglucosaminidase