Beta-galactoside alpha-2,3-sialyltransferase

 

Glycosyltransferase enzymes perform the sialyation of glycojonguates, molecules which have vital roles in cell recognition, adhesion and immunogenicity. The subset of sialyl-transferases (ST) catalyse the transfer of the sialic acid moiety from a cytidine-5'-monophosphate-N-acyl neuraminic acid donor to various acceptor glyco-conjuates terminating in either galactose, N-acetyl-galactosamine or another sialic acid. The enzyme annotated here, ST3Gal-I, catalyses the displacement reaction between CMP-N-acetylneuraminate and beta-D-Galactosyl-1,3-N-acetyl-alpha-D-galactosaminyl and is a member of the glycosyl transferase family 29. Bacterial sialytransferase enzymes contain a DXD domain which binds two divalent cations that are essential for catalysis. No such domain is present in the mammalian homologues, and the activity of these enzymes is found not to be dependent upon the presence of metal cofactors [PMID:19820709].

While the glycosyltransferase-catalysed reactions had been assumed to be uni-directional, the narrow difference in favourability for glycoside formation has been exploited to synthesise rare NDP-sugar products [PMID:16946071].

 

Reference Protein and Structure

Sequence
Q02745 UniProt (2.4.99.2, 2.4.99.4) IPR012163 (Sequence Homologues) (PDB Homologues)
Biological species
Sus scrofa (pig) Uniprot
PDB
2wml - Crystal Structure of a Mammalian Sialyltransferase (1.9 Å) PDBe PDBsum 2wml
Catalytic CATH Domains
3.90.1480.20 CATHdb (see all for 2wml)
Click To Show Structure

Enzyme Reaction (EC:2.4.99.4)

beta-D-galactosyl-(1->3)-N-acetyl-alpha-D-galactosaminyl group
CHEBI:16117ChEBI
+
CMP-N-acetyl-beta-neuraminate(2-)
CHEBI:57812ChEBI
→
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
Alternative enzyme names: CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase, CMP-N-acetylneuraminate:beta-D-galactoside alpha-2,3-N-acetylneuraminyl-transferase,

Enzyme Mechanism

Introduction

His319 activates the hydroxyl of the acceptor group towards nucleophilic attack at the donor sugar (Sn2 reaction). Since the active site has little access to solvent, presumably to reduce unwanted hydrolysis of the substrates or products, it has been inferred that the proton is reincorporated into the products rather than remaining in the active site [PMID:19820709, PMID:21098518].

Catalytic Residues Roles

UniProt PDB* (2wml)
Tyr269 Tyr269(224)A Helps direct and stabilise the transition states of the reaction. hydrogen bond acceptor, steric role
His319 His319(274)A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator
*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

bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used, rate-determining step, inferred reaction step, native state of enzyme regenerated

References

  1. Rao FV et al. (2009), Nat Struct Mol Biol, 16, 1186-1188. Structural insight into mammalian sialyltransferases. DOI:10.1038/nsmb.1685. PMID:19820709.
  2. Bhide GP et al. (2017), Histochem Cell Biol, 147, 149-174. Sialylation of N-glycans: mechanism, cellular compartmentalization and function. DOI:10.1007/s00418-016-1520-x. PMID:27975143.
  3. Audry M et al. (2011), Glycobiology, 21, 716-726. Current trends in the structure-activity relationships of sialyltransferases. DOI:10.1093/glycob/cwq189. PMID:21098518.
  4. Chan PH et al. (2009), Biochemistry, 48, 11220-11230. NMR Spectroscopic Characterization of the Sialyltransferase CstII fromCampylobacter jejuni: Histidine 188 Is the General Base. DOI:10.1021/bi901606n. PMID:19824695.
  5. Zhang C et al. (2006), Science, 313, 1291-1294. Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions. DOI:10.1126/science.1130028. PMID:16946071.

Step 1. His319 activates the hydroxyl of the acceptor group towards nucleophilic attack at the donor sugar.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269(224)A hydrogen bond acceptor, steric role
His319(274)A activator, hydrogen bond acceptor, hydrogen bond donor, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used, rate-determining step

Step 2. It is unclear how the starting state of the histidine is regenerated. Most likely, the leaving CMP abstracts the proton.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His319(274)A proton donor

Chemical Components

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

Step 1. His319 activates the hydroxyl of the acceptor group towards nucleophilic attack at the donor sugar.

Step 2. It is unclear how the starting state of the histidine is regenerated. Most likely, the leaving CMP abstracts the proton.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Morwenna Hall