Exo-alpha-sialidase (GH33 Family)

 

Exo sialidases (neuraminidases) catalyse the removal of terminal sialic acid residues in oligosaccharides, glycoproteins, glycolipids and synthetic substrates to release free sialic acid. This class function to release sialic acids for use as carbon and energy sources for non-pathogenic bacterium while in pathogenic microorganisms, sialidases have been suggested to be pathogenic factors.

 

Reference Protein and Structure

Sequence
Q02834 UniProt (3.2.1.18) IPR026856 (Sequence Homologues) (PDB Homologues)
Biological species
Micromonospora viridifaciens (Bacteria) Uniprot
PDB
1euu - SIALIDASE OR NEURAMINIDASE, LARGE 68KD FORM (2.5 Å) PDBe PDBsum 1euu
Catalytic CATH Domains
2.120.10.10 CATHdb (see all for 1euu)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.18)

water
CHEBI:15377ChEBI
+
alpha-N-acetylneuraminyl-(2->3)-beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1')-ceramide
CHEBI:15681ChEBI
→
N-acetylneuraminic acid
CHEBI:17012ChEBI
+
beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1)-N-acylsphingosine
CHEBI:17950ChEBI
Alternative enzyme names: Alpha-neuraminidase, Acetylneuraminidase, Neuraminidase, Sialidase, N-acylneuraminate glycohydrolase,

Enzyme Mechanism

Introduction

The three catalytic residues are Tyr 370, Glu 260 and Asp 92. Tyr 370 acts as a nucleophile to attack the C1 carbon of sialic acid in an SN2 mechanism, with concerted displacement of the glycoconjagate to which the sialic acid is attached. The departing oxygen atom of the sialyl ketal linkage is protonated by Asp 92. Glu 260 functions to deprotonate Tyr 370 as it attacks the sialic acid. In the second step, Asp 92 deprotates a water molecule that attacks C1 of the intermediate to displace Tyr 370 and generate sialic acid.

Catalytic Residues Roles

UniProt PDB* (1euu)
Asp92 Asp92(50)A Protonates the departing oxygen of the sialyl ketal linkage. Deprotonates the water molecule that attacks the tyrosine-linked intermediate. proton acceptor, proton donor, activator, increase nucleophilicity, promote heterolysis
Tyr370 Tyr370(328)A Attacks the C1 carbon of sialic acid to displace the glycoconjugate in an SN2 mechanism. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
Glu260 Glu260(218)A Deprotonates Tyr 370 which acts as a nucleophile to attack C1 of the sialic acid. proton acceptor, proton donor, activator, increase nucleophilicity, promote heterolysis
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

overall product formed, intermediate formation, overall reactant used, bimolecular nucleophilic substitution, proton transfer, intermediate terminated, native state of enzyme regenerated, hydrolysis

References

  1. Amaya MF et al. (2004), Structure, 12, 775-784. Structural Insights into the Catalytic Mechanism of Trypanosoma cruzi trans-Sialidase. DOI:10.1016/j.str.2004.02.036. PMID:15130470.
  2. Bueren-Calabuig JA et al. (2014), J Phys Chem B, 118, 5807-5816. Unraveling the differences of the hydrolytic activity of Trypanosoma cruzi trans-sialidase and Trypanosoma rangeli sialidase: a quantum mechanics-molecular mechanics modeling study. DOI:10.1021/jp412294r. PMID:24814976.
  3. Oliveira IA et al. (2014), J Biol Chem, 289, 423-436. Evidence of ternary complex formation in Trypanosoma cruzi trans-sialidase catalysis. DOI:10.1074/jbc.M112.399303. PMID:24194520.
  4. Pierdominici-Sottile G et al. (2011), Biochemistry, 50, 10150-10158. Free Energy Study of the Catalytic Mechanism ofTrypanosoma cruzitrans-Sialidase. From the Michaelis Complex to the Covalent Intermediate. DOI:doi:10.1021/bi2009618.
  5. Watson JN et al. (2004), FEBS Lett, 577, 265-269. Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase fromMicromonospora viridifaciens. DOI:10.1016/j.febslet.2004.10.016. PMID:15527797.
  6. Watson JN et al. (2003), Biochemistry, 42, 12682-12690. Mutagenesis of the Conserved Active-Site Tyrosine Changes a Retaining Sialidase into an Inverting Sialidase†. DOI:10.1021/bi035396g. PMID:14580216.
  7. Watts AG et al. (2003), J Am Chem Soc, 125, 7532-7533. Trypanosoma cruziTrans-sialidase Operates through a Covalent Sialyl−Enzyme Intermediate:  Tyrosine Is the Catalytic Nucleophile. DOI:10.1021/ja0344967. PMID:12812490.
  8. Gaskell A et al. (1995), Structure, 3, 1197-1205. The three domains of a bacterial sialidase: a β-propeller, an immunoglobulin module and a galactose-binding jelly-roll. DOI:10.1016/s0969-2126(01)00255-6. PMID:8591030.

Step 1. Glu260 activates Tyr370 which attacks C1. The sialyl ketal linkage is broken in an SN2 reaction with Asp92 protonating the leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr370(328)A covalently attached
Glu260(218)A activator, increase nucleophilicity
Asp92(50)A promote heterolysis
Glu260(218)A proton acceptor
Tyr370(328)A proton donor, nucleophile
Asp92(50)A proton donor

Chemical Components

overall product formed, intermediate formation, overall reactant used, ingold: bimolecular nucleophilic substitution, proton transfer

Step 2. Asp92 activates a water molecule which hydrolyses the tyrosine bound intermediate. Glu260 protonates the departing Tyr370.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu260(218)A promote heterolysis
Asp92(50)A activator, increase nucleophilicity
Tyr370(328)A proton acceptor
Asp92(50)A proton acceptor
Glu260(218)A proton donor
Tyr370(328)A nucleofuge

Chemical Components

overall product formed, intermediate terminated, native state of enzyme regenerated, ingold: bimolecular nucleophilic substitution, proton transfer, hydrolysis
Download: Image, Marvin File

Step 1. Glu260 activates Tyr370 which attacks C1. The sialyl ketal linkage is broken in an SN2 reaction with Asp92 protonating the leaving group.

Step 2. Asp92 activates a water molecule which hydrolyses the tyrosine bound intermediate. Glu260 protonates the departing Tyr370.

Products.

Contributors

Steven Smith, Gemma L. Holliday, James Willey