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. 2012 May;22(5):590-5.
doi: 10.1093/glycob/cwr165. Epub 2011 Nov 16.

Structural analysis of a bacterial exo-α-D-N-acetylglucosaminidase in complex with an unusual disaccharide found in class III mucin

Elizabeth Ficko-Blean  1 , Alisdair B Boraston
Affiliations

Structural analysis of a bacterial exo-α-D-N-acetylglucosaminidase in complex with an unusual disaccharide found in class III mucin

Elizabeth Ficko-Blean et al. Glycobiology. 2012 May.

Abstract

CpGH89 is a family 89 glycoside hydrolase with exo-α-D-N-acetylglucosaminidase activity that is produced by the human and animal pathogen Clostridium perfringens. This enzyme is active on the α-D-GlcpNAc-(1 → 4)-D-Galp motif that is displayed on the class III mucins within the gastric mucosa. Other members of this enzyme family, such as human NAGLU, are active on heparan. A truncated version of CpGH89 was rendered inactive through the mutation of two key catalytic residues, the protein crystallized and its structure determined in complex with α-D-GlcpNAc-(1 → 4)-D-Galp to reveal the molecular details of how this unique disaccharide is recognized by CpGH89. An analysis of this substrate complex not only provides insight into how this enzyme selects for its mucin-presented substrate but also advances our understanding of how its clinically relevant mammalian counterparts are specific for heparan.

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Figures

Fig. 1.
Fig. 1.
Glycan microarray analysis of carbohydrate binding by GH89CMmut. The carbohydrates giving the most significant signals are numbered and schematics of the structures of these glycans are given to the right of the array results. The glycan array number of the oligosaccharide is given to the left of the schematic with the raw fluorescence intensity from the array analysis given in parentheses beneath the glycan number. Dark gray boxes represent N-acetylglucosamine; light gray circles, galactose; dark gray circles, glucose; light gray boxes, N-acetylgalactosamine; triangles, fucose. The errors and error bars represent the standard error of the mean for measurements made in quadruplicate.
Fig. 2.
Fig. 2.
Structural analysis of GH89CMmut in complex with α-d-GlcpNAc-(1 → 4)-d-Galp. (A) Electron density for α-d-GlcpNAc-(1 → 4)-d-Galp in the active site of GH89CMmut. The electron density maps are maximum-likelihood (Murshudov et al. 1997)/σA (Read 1986) − weighted 2Fobs − Fcalc contoured at 1 σ (both at 0.29 e−/Å3) produced with the disaccharide omitted (green) and included (blue) in the refinement. (B) The cutaway view of the GH89CMmut active site surface showing accommodation of the α-d-GlcpNAc-(1 → 4)-d-Galp disaccharides. The mutated acid/base residue, Q483, and mutated nucleophile, Q601, are shown in stick representation with relevant distances shown as dashed lines and labeled. The subsites are labeled according to the convention established by Davies et al. (1997). (C) The divergent stereo view of the primary apolar interactions in the active site. (D) Schematic of the hydrogen-bonding pattern between the enzyme active site and the disaccharide substrate. (E) Conservation of functional residues determined using CONSURF (Ashkenazy et al. 2010) focusing on the active site of CpGH89. One hundred and forty-three unique amino acid sequences having greater than 28% amino acid sequence identity with the catalytic module of CpGH89 were used in the analysis. The color-coding scale for conservation is given immediately below the panel. (F) Sequence alignments of GH89, three randomly chosen bacterial examples of GH89 from Bacteroides vulgatus (BvGH89), Bifidobacterium bifidum (BbGH89) and Streptomyces avermitilis (SaGH89), and three mammalian GH89 enzymes from Homo sapiens (HsGH89; also referred to as NAGLU), Mus musculus (MmGH89) and Bos Taurus (BtGH89). These aligned segments show regions that correspond to the +1 subsite. Key residues involved in galactose recognition in the +1 subsite of CpGH89 are indicated with triangles and color coded to show hydrophobic interaction (purple) or hydrogen bonding (yellow). (G) The catalytic site of GH89CMmut superposed with a homology model of human NAGLU. The solvent accessible surface of human NAGLU is shown with the α-d-GlcpNAc-(1 → 4)-d-Galp disaccharide (blue sticks) from the GH89CMmut structure in the active site. W685 is shown in pink stick representation. (H) An overlay of α-d-GlcpNAc-(1 → 4)-d-Galp (blue) from the GH89CMmut structure with the GLYCAM (Kirschner et al. 2008)-modeled α-d-GlcpNAc-(1 → 4)-d-GlcAp (green) disaccharide present in heparan. W685, which is a key determinant of galactose recognition the +1 subsite, is shown in stick representation.
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