Endo-alpha-sialidase

 

E. coli K1, a major cause of neonatal sepsis and meningitis, surrounds itself in a capsule of poly alpha2,8-sialic acid (polySia), a sugar polymer that also acts as an important modulator of neuronal plasticity in the adult human host; hence, the mammalian immune system does not attack the bacterium. The bacteriophage K1F can hydrolyse this bacterial capsule using an endosialidase, which is a mushroom-shaped homotrimer. This enzyme may find use in the diagnosis or therapy of polySia-bearing tumours, and in the treatment of meningitis caused by polySia-encapsulated bacteria.

 

Reference Protein and Structure

Sequence
Q04830 UniProt (3.2.1.129) IPR001724 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage K1F (Virus) Uniprot
PDB
1v0e - Endosialidase of Bacteriophage K1F (1.9 Å) PDBe PDBsum 1v0e
Catalytic CATH Domains
2.120.10.10 CATHdb (see all for 1v0e)

Enzyme Reaction (EC:3.2.1.129)

water
CHEBI:15377ChEBI
+
alpha-Neup5Ac-(2->8)-alpha-Neup5Ac
CHEBI:62097ChEBI
→
N-acetylneuraminate
CHEBI:35418ChEBI
+
N-acetylneuraminate
CHEBI:35418ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Endo-N-acylneuraminidase, Alpha-2,8-sialosylhydrolase, Endo-N-acetylneuraminidase, Endoneuraminidase, Endosialidase, Poly(alpha-2,8-sialoside) alpha-2,8-sialosylhydrolase, Poly(alpha-2,8-sialosyl) endo-N-acetylneuraminidase,

Enzyme Mechanism

Introduction

Endosialidases are proposed to work by catalysing the intramolecular self-cleavage of polySia that occurs spontaneously in mild acidic conditions: Arg 596 and Arg 647 bind the carboxyl group of polySia. This raises the pKa of the carboxyl so that it is protonated. The binding may also force the pyranose ring into an unfavourable boat conformation. The proton is transferred (intramolecularly) between the carboxyl and the oxygen atom in the glycosidic linkage, making the linking oxygen a good leaving group. The lone pair on the ring oxygen forms a -C=O+- bond, triggering the cleavage of the glycosidic bond and creating a planar, cationic transition state and intermediate that is stabilised by Glu 581. Water is nucleophilic and attacks the carbon of the -C=O+- group, quenching the positive charge on oxygen and relieving the strained planar conformation of the intermediate. Water is activated by Glu581. The products formed have terminal sialic acid units, one a hemiacetal and the other a primary alcohol.

Catalytic Residues Roles

UniProt PDB* (1v0e)
Glu581 Glu581(337)A Stabilises the planar cationic transition states and intermediate. Activates the water for nucleophilic attack. proton acceptor, increase nucleophilicity, activator, electrostatic stabiliser
Arg596 Arg596(352)A Arg 596 increases the pKa of the carboxylate group of the substrate and effects the change of pyranose conformation. electrostatic stabiliser, increase acidity
Arg647 Arg647(403)A Arg 647 increases the pKa of the carboxylate group of the substrate and effects the change of pyranose conformation. electrostatic stabiliser, increase acidity
*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

proton transfer, overall reactant used, electron transfer, heterolysis, overall product formed, bimolecular nucleophilic addition

References

  1. Stummeyer K et al. (2005), Nat Struct Mol Biol, 12, 90-96. Crystal structure of the polysialic acid–degrading endosialidase of bacteriophage K1F. DOI:10.1038/nsmb874. PMID:15608653.
  2. Manzi AE et al. (1994), J Biol Chem, 269, 23617-23624. Intramolecular self-cleavage of polysialic acid. PMID:8089131.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, James Willey