Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae.
Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2(1) and diffracted to 2.0 A resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 A, beta = 112.46 degrees. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-alpha-D-galactopyranoside, which is consistent with the classification of this enzyme as an endo-alpha-N-acetylgalactosaminidase.
Gregg
Katie J
KJ
Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, British Columbia V8W 3P6, Canada.
Boraston
Alisdair B
AB
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics*; D040681:Structural Homology, Protein; D014961:X-Ray Diffraction / Q000379:methods; D048809:alpha-N-Acetylgalactosaminidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D048809:alpha-N-Acetylgalactosaminidase