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384 a.a.
_ZN
Key reference
DOI no: 10.1073/pnas.0504339102 Proc Natl Acad Sci U S A 102:15429-15434 (2005) PubMed id: 16221761
Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor. D.E.Blair, A.W.Schüttelkopf, J.I.MacRae, D.M.van Aalten. ABSTRACT
Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc(3) as a substrate (K(m) = 3.8 mM, k(cat) = 0.55 s(-1)), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanism-based inhibitors for this important class of enzymes.
Selected figure(s)
Figure 2.
Fig. 2. Details of the SpPgdA active site. Close-up of the active sites of: the native SpPgdA structure in complex with the acetate product and PEG200 (SpPGDA_Ac), the SpPgdA D275N mutant in complex with sulfate and Mes (SpPGDA_SO4), and the previously determined complex of B. subtilis PdaA in complex with GlcNAc and a glycerol molecule (PDAA_GlcNAc). The five CE-4 sequence motifs (MT1-5) are shown in yellow. Side chains lining the active site cleft are shown as sticks. Residues conserved in all CE-4 esterases are magenta. Water molecules (spheres) and ligands (green sticks) are also shown. Unbiased F[o] - F[c],[calc] maps are shown at 2.25
(Mes in SpPGDA_SO4) and 12
Figure 4.
Fig. 4. Docked GlcNAc[3] complex and reaction mechanism. (A) Sp-PgdA is shown as a ribbon, with conserved side chains. The acetate molecule as observed in the SpPgdA-acetate complex (Fig. 2) is shown as yellow sticks. GlcNAc[3] is shown as green sticks with subsites labeled in blue. Hydrogen bonds are shown as dashed lines in black, and zinc-ligand interactions are shown as magenta dashed lines. (B) As A, but viewed from the top. (C) Proposed catalytic mechanism.Figures were selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
18978064 D.M.Deng, J.E.Urch, J.M.ten Cate, V.A.Rao, D.M.van Aalten, and W.Crielaard (2009).
Streptococcus mutans SMU.623c codes for a functional, metal-dependent polysaccharide deacetylase that modulates interactions with salivary agglutinin.J Bacteriol, 191, 394-402.
PDB code: 2w3z
19472335 J.E.Urch, R.Hurtado-Guerrero, D.Brosson, Z.Liu, V.G.Eijsink, C.Texier, and D.M.van Aalten (2009).
Structural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculi.Protein Sci, 18, 1197-1209.
18990186 N.Fittipaldi, T.Sekizaki, D.Takamatsu, M.d.e. .L.Domínguez-Punaro, J.Harel, N.K.Bui, W.Vollmer, and M.Gottschalk (2008).
Significant contribution of the pgdA gene to the virulence of Streptococcus suis.Mol Microbiol, 70, 1120-1135.
18828843 U.Toprak, D.Baldwin, M.Erlandson, C.Gillott, X.Hou, C.Coutu, and D.D.Hegedus (2008).
A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix.Insect Mol Biol, 17, 573-585.
18070068 W.Vollmer (2008).
Structural variation in the glycan strands of bacterial peptidoglycan.FEMS Microbiol Rev, 32, 287-306.
17637984 D.P.Giedroc, and A.I.Arunkumar (2007).
Metal sensor proteins: nature's metalloregulated allosteric switches.Dalton Trans, 0, 3107-3120.
17785473 L.Hébert, P.Courtin, R.Torelli, M.Sanguinetti, M.P.Chapot-Chartier, Y.Auffray, and A.Benachour (2007).
Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance.Infect Immun, 75, 5390-5398.
17063474 L.Oberbarnscheidt, E.J.Taylor, G.J.Davies, and T.M.Gloster (2007).
Structure of a carbohydrate esterase from Bacillus anthracis.Proteins, 66, 250-252.
PDB code: 2j13
17327397 P.Mercier, M.J.Lewis, D.D.Hau, L.F.Saltibus, W.Xiao, and L.Spyracopoulos (2007).
Structure, interactions, and dynamics of the RING domain from human TRAF6.Protein Sci, 16, 602-614.
PDB code: 2jmd
16672610 T.Chitlaru, O.Gat, Y.Gozlan, N.Ariel, and A.Shafferman (2006).
Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities.J Bacteriol, 188, 3551-3571. The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.
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