Alpha-2,3-sialyltransferase

 

Sialyltransferase (CstII) from Campylobacter jejuni catalyses the transfer of the sialic acid moiety from cytidine-5'-monophospho-N-acetyl-neuramic acid (CMP-NeuAc) to the terminal positions of glycoconjugates. The products play critical roles in recognition and adherence. Campylobacter jejuni has been shown to express variable cell surface carbohydrate mimics of human gangliosides that are associated with virulence.

 

Reference Protein and Structure

Sequence
Q9LAK3 UniProt IPR009251 (Sequence Homologues) (PDB Homologues)
Biological species
Campylobacter jejuni (Bacteria) Uniprot
PDB
1ro7 - Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analogue, CMP-3FNeuAc. (1.8 Å) PDBe PDBsum 1ro7
Catalytic CATH Domains
3.90.1480.10 CATHdb (see all for 1ro7)
Click To Show Structure

Enzyme Reaction (EC:2.4.99.-)

CMP-N-acetyl-beta-neuraminate(2-)
CHEBI:57812ChEBI
+
beta-D-galactosyl-(1->3)-N-acetyl-alpha-D-galactosaminyl group
CHEBI:16117ChEBI
→
alpha-N-acetylneuraminyl-2,3-beta-D-galactosyl-1,3-N-acetyl-alpha-D-galactosaminyl group(1-)
CHEBI:60068ChEBI
+
cytidine 5'-monophosphate(2-)
CHEBI:60377ChEBI
+
hydron
CHEBI:15378ChEBI

Enzyme Mechanism

Introduction

Unlike other inverting glucosyltransferases, the reaction proceeds in an Sn1 manner, initiated by elimination of CMP (promoted by Tyr156 and Tyr162). His 188 is stabilised in its unprotonated state by Arg 129. His 188 accepts a proton from the O3' hydroxyl, activating it for nucleophilic attack on the donor C2' atom.

Catalytic Residues Roles

UniProt PDB* (1ro7)
Arg129 Arg129A Acts to provide an electrostatic shield favouring the deprotonated form of His188. electrostatic stabiliser
Tyr156, Tyr162 Tyr156A, Tyr162A Stabilises the phosphate leaving group by hydrogen bonding to the non-bridging, pro-S oxygen which help prevents the reverse reaction. electrostatic stabiliser
His188 His188A His 188 acts as a general base by deprotonating the O3' hydroxyl of the acceptor galactose, activating it for nucleophilic attack at the anomeric carbon of the donor sugar. activator, proton acceptor, proton donor
*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

heterolysis, intermediate formation, elimination (not covered by the Ingold mechanisms), overall reactant used, bimolecular nucleophilic addition, proton transfer, intermediate terminated, overall product formed, native state of enzyme regenerated

References

  1. Hamada Y et al. (2016), Biochemistry, 55, 5764-5771. Quantum Mechanics/Molecular Mechanics Study of the Sialyltransferase Reaction Mechanism. DOI:10.1021/acs.biochem.6b00267. PMID:27644888.
  2. Chiu CP et al. (2004), Nat Struct Mol Biol, 11, 163-170. Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. DOI:10.1038/nsmb720. PMID:14730352.

Step 1. Cleavage of C2-O glycosidic bond, stabilised by Tyr156 and Tyr162.

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Catalytic Residues Roles

Residue Roles
Tyr156A electrostatic stabiliser
Arg129A electrostatic stabiliser
Tyr162A electrostatic stabiliser

Chemical Components

heterolysis, intermediate formation, elimination (not covered by the Ingold mechanisms), overall reactant used

Step 2. His188 deprotonates the 3′-OH of galactose, increasing it's nucleophilicity to then attack the oxocarbenium ion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His188A activator
His188A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, intermediate terminated, overall product formed

Step 3. Proton transfer from His188 to CMP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His188A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Cleavage of C2-O glycosidic bond, stabilised by Tyr156 and Tyr162.

Step 2. His188 deprotonates the 3′-OH of galactose, increasing it's nucleophilicity to then attack the oxocarbenium ion intermediate.

Step 3. Proton transfer from His188 to CMP.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Morwenna Hall