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)
Click To Show Structure

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.

Step 1. There is an intramolecular proton transfer from the carboxylate group to the oxygen of the glycosidic linkage.

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Catalytic Residues Roles

Residue Roles
Arg596(352)A electrostatic stabiliser
Arg647(403)A electrostatic stabiliser
Arg596(352)A increase acidity
Arg647(403)A increase acidity
Glu581(337)A electrostatic stabiliser

Chemical Components

proton transfer, overall reactant used

Step 2. Electron movement from the ring oxygen to form a C=O bond causes the cleavage of the glycosidic linkage.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg596(352)A electrostatic stabiliser
Arg647(403)A electrostatic stabiliser
Glu581(337)A electrostatic stabiliser

Chemical Components

electron transfer, heterolysis, overall product formed

Step 3. Glu581 activates a water molecule for nucleophilic attack on the C=O bond and the product is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu581(337)A electrostatic stabiliser
Arg596(352)A electrostatic stabiliser
Arg647(403)A electrostatic stabiliser
Glu581(337)A increase nucleophilicity, activator, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall product formed
Download: Image, Marvin File

Step 1. There is an intramolecular proton transfer from the carboxylate group to the oxygen of the glycosidic linkage.

Step 2. Electron movement from the ring oxygen to form a C=O bond causes the cleavage of the glycosidic linkage.

Step 3. Glu581 activates a water molecule for nucleophilic attack on the C=O bond and the product is formed.

Products.

Introduction

In this mechanism Glu581 activates a water molecule for nucleophilic attack on the glycosidic linkage. The leaving group is protonated by the His350/Tyr325 proton relay. The two arginine residues act as electrostatic stabilizers.

Catalytic Residues Roles

UniProt PDB* (1v0e)
Glu581 Glu581(337)A Activates the water molecule for nucleophilic attack. proton acceptor, proton donor, activator, electrostatic stabiliser, increase nucleophilicity
His350, Tyr325 His350(106)A, Tyr325(81)A Part of the proton relay which protonates the leaving group. proton relay, promote heterolysis, proton acceptor, proton donor
Arg596, Arg647 Arg596(352)A, Arg647(403)A Electrostatic stabilizers. electrostatic stabiliser
*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, proton relay, overall product formed, overall reactant used, bimolecular nucleophilic substitution, native state of enzyme regenerated

References

  1. Jakobsson E et al. (2015), Top Curr Chem, 367, 29-73. Endosialidases: Versatile Tools for the Study of Polysialic Acid. DOI:10.1007/128_2012_349. PMID:22851159.

Step 1. Glu581 activates a water molecule for nucleophilic attack on the glycosidic linkage. The His350/Tyr325 proton relay donates a proton to the leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu581(337)A electrostatic stabiliser
Arg596(352)A electrostatic stabiliser
Arg647(403)A electrostatic stabiliser
His350(106)A proton relay
Glu581(337)A increase nucleophilicity
Tyr325(81)A promote heterolysis
His350(106)A promote heterolysis
Glu581(337)A activator, proton acceptor
His350(106)A proton acceptor
Tyr325(81)A proton donor
His350(106)A proton donor

Chemical Components

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

Step 2. The proton relay regenerates the active site via a number of water molecules.

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Catalytic Residues Roles

Residue Roles
His350(106)A proton relay, proton acceptor
Tyr325(81)A proton acceptor
His350(106)A proton donor
Glu581(337)A proton donor

Chemical Components

proton relay, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu581 activates a water molecule for nucleophilic attack on the glycosidic linkage. The His350/Tyr325 proton relay donates a proton to the leaving group.

Step 2. The proton relay regenerates the active site via a number of water molecules.

Products.

Introduction

This is a single step version of the mechanism where the base is the substrate's own carboxylate group; this activates a water molecule which attacks the glycosidic linkage. Glu581 acts as an acid donating a proton to the leaving group. This mechanism follows an SN2 scheme.

Catalytic Residues Roles

UniProt PDB* (1v0e)
Glu581 Glu581(337)A Donates a proton to the leaving group. promote heterolysis, proton donor
Arg596, Arg647 Arg596(352)A, Arg647(403)A Stabilize the carboxylate group. electrostatic stabiliser
*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 product formed, overall reactant used, bimolecular nucleophilic substitution

References

  1. Morley TJ et al. (2009), J Biol Chem, 284, 17404-17410. A new sialidase mechanism: bacteriophage K1F endo-sialidase is an inverting glycosidase. DOI:10.1074/jbc.M109.003970. PMID:19411257.

Step 1. The carboxylate group of the substrate acts as a base to deprotonate a water molecule which performs nucleophilic attack on the glycosidic linkage. Glu581 acts an acid donating a proton to the leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg596(352)A electrostatic stabiliser
Arg647(403)A electrostatic stabiliser
Glu581(337)A promote heterolysis, proton donor

Chemical Components

proton transfer, overall product formed, overall reactant used, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. The carboxylate group of the substrate acts as a base to deprotonate a water molecule which performs nucleophilic attack on the glycosidic linkage. Glu581 acts an acid donating a proton to the leaving group.

Products.

Contributors

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