Β-N-acetylglucosaminylglycopeptide β-1,4-galactosyltransferase

 

Beta-1,4-galactosyltransferase-1 (Gal-T1) is a Golgi resident type II protein that catalyses the transfer of galactose from UDP-Gal to N-acetylglucosamine in the presence of Mg2+, forming the product N-acetyl-lactosamine. The 1-4- galactosyltransferase (Gal-T) family, consists of at least seven members, including Gal-T1, with a 25 to 55% sequence homology. These enzymes are expressed in different tissues and show differences in the oligosaccharide acceptor specificity.

 

Reference Protein and Structure

Sequence
P08037 UniProt (2.4.1.-, 2.4.1.22, 2.4.1.38, 2.4.1.90, 2.4.1.275) IPR003859 (Sequence Homologues) (PDB Homologues)
Biological species
Bos taurus (Cattle) Uniprot
PDB
1fr8 - CRYSTAL STRUCTURE OF THE BOVINE BETA 1,4 GALACTOSYLTRANSFERASE (B4GALT1) CATALYTIC DOMAIN COMPLEXED WITH URIDINE DIPHOSPHOGALACTOSE (2.4 Å) PDBe PDBsum 1fr8
Catalytic CATH Domains
3.90.550.10 CATHdb (see all for 1fr8)
Cofactors
Manganese(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.38)

N-acetyl-beta-D-glucosaminide
CHEBI:61631ChEBI
+
UDP-alpha-D-galactose(2-)
CHEBI:66914ChEBI
→
beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminide
CHEBI:133507ChEBI
+
UDP(3-)
CHEBI:58223ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Beta-N-acetyl-beta-1-4-galactosyltransferase, UDPgalactose-glycoprotein galactosyltransferase, UDPgalactose:N-acetyl-beta-D-glucosaminylglycopeptide beta-1,4-galactosyltransferase, Glycoprotein 4-beta-galactosyl-transferase, Glycoprotein beta-galactosyltransferase, Thyroid galactosyltransferase, Thyroid glycoprotein beta-galactosyltransferase, Uridine diphosphogalactose-glycoprotein galactosyltransferase, UDP-galactose--glycoprotein galactosyltransferase, Glycoprotein 4-beta-galactosyltransferase, GalT, UDP-galactose:N-acetyl-beta-D-glucosaminylglycopeptide beta-1,4-galactosyltransferase, UDP-galactose:N-acetyl-beta-D-glucosaminylglycopeptide 4-beta-galactosyltransferase,

Enzyme Mechanism

Introduction

The reaction proceeds in an SN2 type mechanism via the formation of an oxocarbenium ion-like transition state. As the acceptor glucose molecule binds to the enzyme-Mn(II)-UDP-Gal-N-acetylglucosamine complex, the O4 atoms of the glucose forms a strong hydrogen bond with the side chain carboxylate group of Asp318. Due to the presence of the negatively charged carboxylate group, the O4 hydroxyl hydrogen atom is stripped from the acceptor sugar, creating a negative charge on the O4 oxygen. A conformation change in the Gal-N- acetylglucosamine moiety from 4-C-1 to 4-H-3, induced by the developing charge on O4, initiates cleavage of the Gal-N- acetylglucosamine unit form UDP-Gal-N- acetylglucosamine. This elimination is facilitated by the divalent Mn cation which neutralises the negative charge on the UDP. The change in puckering positions the positively charged C1 atom of the Gal-N- acetylglucosamine moiety in-line and in close proximity with the negatively charged O4 oxygen of the sugar acceptor, thus activating the formation of the disaccaride unit.

Catalytic Residues Roles

UniProt PDB* (1fr8)
Arg349, Trp314 Arg349(235)A, Trp314(200)A Stabilise the developing negative charges on the pyrophosphate during the reaction by becoming hydrogen bond donors. hydrogen bond donor, electrostatic stabiliser
Met344, His347, Asp254 Met344(230)A, His347(233)A, Asp254(140)A Co-ordinate to Mn2+ ion which in turn helps promote transition state formation. metal ligand
Asp252 Asp252(138)A The negatively charged side chain of Asp252 helps to stabilise the oxocarbenium transition state. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Glu317 Glu317(203)A The negatively charged side chain of Glu317 helps to stabilise the oxocarbenium transition state. hydrogen bond acceptor, electrostatic stabiliser
Asp318 Asp318(204)A The proton of the O4 hydroxyl group of the sugar acceptor molecule forms a hydrogen bond to, and is deprotonated by Asp318. This increases the substrate's nucleophilicity and also acts to neutralise, and therefore stabilise the oxocarbenium transition state proton acceptor, activator, electrostatic stabiliser, 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

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

References

  1. Ramakrishnan B et al. (2006), J Mol Biol, 357, 1619-1633. Structural Snapshots of β-1,4-Galactosyltransferase-I Along the Kinetic Pathway. DOI:10.1016/j.jmb.2006.01.088. PMID:16497331.
  2. Krupicka M et al. (2009), J Phys Chem B, 113, 11314-11319. Hybrid quantum mechanical/molecular mechanical investigation of the beta-1,4-galactosyltransferase-I mechanism. DOI:10.1021/jp904716t. PMID:19627105.
  3. Ramakrishnan B et al. (2004), Curr Opin Struct Biol, 14, 593-600. Structure and catalytic cycle of β-1,4-galactosyltransferase. DOI:10.1016/j.sbi.2004.09.006. PMID:15465321.

Step 1. Asp318 acts as a general base and abstracts a proton from the O4 hydroxyl, resulting in an increase in its nucleophilicity to attack C1 on UDP-galactose.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp318(204)A electrostatic stabiliser
Glu317(203)A electrostatic stabiliser
Asp252(138)A electrostatic stabiliser
Asp254(140)A metal ligand
Met344(230)A metal ligand
His347(233)A metal ligand
Glu317(203)A hydrogen bond acceptor
Asp252(138)A hydrogen bond acceptor, hydrogen bond donor
Trp314(200)A hydrogen bond donor
Arg349(235)A hydrogen bond donor
Trp314(200)A electrostatic stabiliser
Arg349(235)A electrostatic stabiliser
Asp318(204)A activator, proton acceptor

Chemical Components

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

Step 2. Asp318 is deprotonated by a water molecule to regenerate active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp318(204)A proton donor

Chemical Components

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

Step 1. Asp318 acts as a general base and abstracts a proton from the O4 hydroxyl, resulting in an increase in its nucleophilicity to attack C1 on UDP-galactose.

Step 2. Asp318 is deprotonated by a water molecule to regenerate active site.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday, Morwenna Hall