PDBsum entry 2fhr

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protein ligands links
Hydrolase PDB id
Protein chain
625 a.a. *
SO4 ×2
Waters ×388
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Trypanosoma rangeli sialidase in complex with 2,3- difluoros (covalent intermediate)
Structure: Sialidase. Chain: a. Engineered: yes
Source: Trypanosoma rangeli. Organism_taxid: 5698. Expressed in: escherichia coli. Expression_system_taxid: 562.
2.20Å     R-factor:   0.164     R-free:   0.232
Authors: M.F.Amaya,P.M.Alzari,A.Buschiazzo
Key ref:
A.G.Watts et al. (2006). Structural and Kinetic Analysis of Two Covalent Sialosyl-Enzyme Intermediates on Trypanosoma rangeli Sialidase. J Biol Chem, 281, 4149-4155. PubMed id: 16298994 DOI: 10.1074/jbc.M510677200
26-Dec-05     Release date:   17-Jan-06    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
O44049  (O44049_TRYRA) -  Sialidase
660 a.a.
625 a.a.*
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Exo-alpha-sialidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     metabolic process   2 terms 
  Biochemical function     exo-alpha-(2->3)-sialidase activity     6 terms  


DOI no: 10.1074/jbc.M510677200 J Biol Chem 281:4149-4155 (2006)
PubMed id: 16298994  
Structural and Kinetic Analysis of Two Covalent Sialosyl-Enzyme Intermediates on Trypanosoma rangeli Sialidase.
A.G.Watts, P.Oppezzo, S.G.Withers, P.M.Alzari, A.Buschiazzo.
Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7A resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to "tune" the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.
  Selected figure(s)  
Figure 1.
A, structures of the fluorinated sialic acid derivatives 2,3-difluoro-N-acetyl-neuraminic acid (1) and 2,3-difluoro-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (2) used as mechanism-based inactivators. B, a typical glycosidase-catalyzed reaction showing glycosylation (k[1]) and deglycosylation (k[2]) rate constants affected by the mechanism-based inactivators.
Figure 5.
3-Fluoro-KDN covalent complex with TrSA. Methionine 96 is observed in three alternate conformations, partially filling the cavity left by the absent N-acetyl group on the sialyl moiety. In the unbound enzyme (PDB code 1N1T) or bound to N-acetyl containing sialic acid derivatives, this Met is observed only in one conformation (corresponding to conformer A in this structure). The water network changes are also highlighted; W360 and W369 interact with important residues in the site. The refined 2mF[o] – DF[c] map contoured at 1σ is shown.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 4149-4155) copyright 2006.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21314964 E.M.Vilei, A.Johansson, Y.Schlatter, K.Redhead, and J.Frey (2011).
Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei.
  Vet Res, 42, 2.  
20511247 A.Albohy, M.D.Li, R.B.Zheng, C.Zou, and C.W.Cairo (2010).
Insight into substrate recognition and catalysis by the human neuraminidase 3 (NEU3) through molecular modeling and site-directed mutagenesis.
  Glycobiology, 20, 1127-1138.  
20695427 A.J.Oakley, S.Barrett, T.S.Peat, J.Newman, V.A.Streltsov, L.Waddington, T.Saito, M.Tashiro, and J.L.McKimm-Breschkin (2010).
Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses.
  J Med Chem, 53, 6421-6431.
PDB codes: 3k36 3k37 3k38 3k39 3k3a
  20944218 A.Kumar, A.Lomize, K.K.Jin, D.Carlton, M.D.Miller, L.Jaroszewski, P.Abdubek, T.Astakhova, H.L.Axelrod, H.J.Chiu, T.Clayton, D.Das, M.C.Deller, L.Duan, J.Feuerhelm, J.C.Grant, A.Grzechnik, G.W.Han, H.E.Klock, M.W.Knuth, P.Kozbial, S.S.Krishna, D.Marciano, D.McMullan, A.T.Morse, E.Nigoghossian, L.Okach, R.Reyes, C.L.Rife, N.Sefcovic, H.J.Tien, C.B.Trame, H.van den Bedem, D.Weekes, Q.Xu, K.O.Hodgson, J.Wooley, M.A.Elsliger, A.M.Deacon, A.Godzik, S.A.Lesley, and I.A.Wilson (2010).
Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 66, 1245-1253.
PDB codes: 2ook 2q3l
20124697 E.C.Schulz, P.Neumann, R.Gerardy-Schahn, G.M.Sheldrick, and R.Ficner (2010).
Structure analysis of endosialidase NF at 0.98 A resolution.
  Acta Crystallogr D Biol Crystallogr, 66, 176-180.
PDB code: 3ju4
20418878 J.Chan, A.R.Lewis, M.Gilbert, M.F.Karwaski, and A.J.Bennet (2010).
A direct NMR method for the measurement of competitive kinetic isotope effects.
  Nat Chem Biol, 6, 405-407.  
20552664 T.V.Vuong, and D.B.Wilson (2010).
Glycoside hydrolases: catalytic base/nucleophile diversity.
  Biotechnol Bioeng, 107, 195-205.  
19216574 O.Demir, and A.E.Roitberg (2009).
Modulation of catalytic function by differential plasticity of the active site: case study of Trypanosoma cruzi trans-sialidase and Trypanosoma rangeli sialidase.
  Biochemistry, 48, 3398-3406.  
18625334 A.Buschiazzo, and P.M.Alzari (2008).
Structural insights into sialic acid enzymology.
  Curr Opin Chem Biol, 12, 565-572.  
  19727327 D.B.Berkowitz, K.R.Karukurichi, la Salud-Bea, D.L.Nelson, and C.D.McCune (2008).
Use of Fluorinated Functionality in Enzyme Inhibitor Development: Mechanistic and Analytical Advantages.
  J Fluor Chem, 129, 731-742.  
18558099 D.J.Vocadlo, and G.J.Davies (2008).
Mechanistic insights into glycosidase chemistry.
  Curr Opin Chem Biol, 12, 539-555.  
  18765901 G.Xu, X.Li, P.W.Andrew, and G.L.Taylor (2008).
Structure of the catalytic domain of Streptococcus pneumoniae sialidase NanA.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 64, 772-775.
PDB codes: 2vvz 2w20
18218621 S.L.Newstead, J.A.Potter, J.C.Wilson, G.Xu, C.H.Chien, A.G.Watts, S.G.Withers, and G.L.Taylor (2008).
The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.
  J Biol Chem, 283, 9080-9088.
PDB codes: 2bf6 2vk5 2vk6 2vk7
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 codes are shown on the right.