Alpha-1,3-galactosetransferase

 

alpha-1,3-galactosetransferase catalyses the transfer of galactose from UDP-alpha-D-galactose into an alpha-1,3 linkage with beta-galactosyl groups in glycoconjugates. It belongs to the family of metal-dependent retaining glycosyltransferase. It is involved in the post translational modification glycosylation of proteins, occurring within the Golgi appararatus.

The enzyme is found in many mammals, including most primates and New World monkeys but is not active in Old World primates. In this group, which also includes humans, the enzyme is inactivated by a frame shift mutation. Therefore, the product, alpha-galactose epitope, is the target of a large fraction of natural antibodies. Many mammals used for xenotranlation of organs express the alpha-galactose epitope product, and without inhibition of alpha-1, 3-galactosetransferase in the donor animal, the presence of antibodies specific to this epitope signal in the recipient human for an immune response towards the organ, resulting in rejection.

 

Reference Protein and Structure

Sequence
P14769 UniProt (2.4.1.87) IPR005076 (Sequence Homologues) (PDB Homologues)
Biological species
Bos taurus (Cattle) Uniprot
PDB
1vzx - Roles of active site tryptophans in substrate binding and catalysis by ALPHA-1,3 GALACTOSYLTRANSFERASE (1.97 Å) PDBe PDBsum 1vzx
Catalytic CATH Domains
3.90.550.10 CATHdb (see all for 1vzx)
Cofactors
Manganese(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.87)

UDP-alpha-D-galactose(2-)
CHEBI:66914ChEBI
+
beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl group
CHEBI:12357ChEBI
→
hydron
CHEBI:15378ChEBI
+
UDP(3-)
CHEBI:58223ChEBI
+
alpha-D-Galp-(1->3)-beta-D-Galp-(1->4)-D-GlcpNAc-yl group
CHEBI:17785ChEBI
Alternative enzyme names: Alpha-galactosyltransferase, Beta-D-galactosyl-N-acetylglucosaminylglycopeptide alpha-1,3-galactosyltransferase, UDP-Gal:N-acetyllactosaminide alpha(1,3)-galactosyltransferase, UDP-Gal:N-acetyllactosaminide alpha-1,3-D-galactosyltransferase, UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase, UDP-Gal:Gal-beta-1->4GlcNAc-R alpha-1->3-galactosyltransferase, UDP-galactose-acetyllactosamine alpha-D-galactosyltransferase, UDPgalactose:beta-D-galactosyl-beta-1,4-N-acetyl-D-glucosaminyl-glycopeptide alpha-1,3-D-galactosyltransferase, Glucosaminylglycopeptide alpha-1,3-galactosyltransferase, Uridine diphosphogalactose-acetyllactosamine alpha-1->3-galactosyltransferase, Uridine diphosphogalactose-acetyllactosamine galactosyltransferase, Uridine diphosphogalactose-galactosylacetylglucosaminylgalactosylglucosylceramide galactosyltransferase, N-acetyllactosaminide alpha-1,3-galactosyltransferase, Galactosyltransferase, Uridine diphosphogalactose-galactosylacetylglucosaminylgalactosyl-glucosylceramide galactosyltransferase, UDP-galactose:N-acetyllactosaminide 3-alpha-D-galactosyltransferase, UDP-galactose:beta-D-galactosyl-1,4-beta-N-acetyl-D-glucosaminyl-R 3-alpha-D-galactosyltransferase, UDP-galactose:beta-D-galactosyl-(1->4)-beta-N-acetyl-D-glucosaminyl-R 3-alpha-D-galactosyltransferase,

Enzyme Mechanism

Introduction

The mechanism follows a single step SNi reaction, in which hydrolysis of UDP-galactose occurs to form a galactose with an oxycarbenium ion character, which is stabilised by Glu317. The transition state is further stabilised by Arg365, Trp314 and Manganese ion. The galactose is transferred to the acceptor substrate by a nucleophilic attack of the hydroxyl group of its anomeric carbon to the oxycarbenium ion.

Catalytic Residues Roles

UniProt PDB* (1vzx)
Glu317 Glu317(238)A Sabilises the cationic transition state with oxocarbenium ion character activator, electrostatic stabiliser
Trp314 Tyr314(235)A Stabilises the transition state in the galactose transfer hydrogen bond donor, electrostatic stabiliser
Trp356, Gln247, His280, Arg365 Trp356(277)A, Gln247(168)A, His280(201)A, Arg365(286)A Stabilises the transition state of the reaction. hydrogen bond donor, 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

overall reactant used, overall product formed, proton transfer, unimolecular elimination by the conjugate base, bimolecular nucleophilic addition

References

  1. Zhang Y et al. (2003), Biochemistry, 42, 13512-13521. Roles of Individual Enzyme−Substrate Interactions by α-1,3-Galactosyltransferase in Catalysis and Specificity†,‡. DOI:10.1021/bi035430r. PMID:14621997.
  2. Gómez H et al. (2013), J Am Chem Soc, 135, 7053-7063. Substrate-assisted and nucleophilically assisted catalysis in bovine α1,3-galactosyltransferase. Mechanistic implications for retaining glycosyltransferases. DOI:10.1021/ja4024447. PMID:23578032.
  3. Jamaluddin H et al. (2009), Biochem Biophys Res Commun, 385, 601-604. Crystal structure of α-1,3-galactosyltransferase (α3GT) in a complex with p-nitrophenyl-β-galactoside (pNPβGal). DOI:10.1016/j.bbrc.2009.05.111. PMID:19486884.
  4. Jamaluddin H et al. (2007), J Mol Biol, 369, 1270-1281. Conformational Changes Induced by Binding UDP-2F-galactose to α-1,3 Galactosyltransferase- Implications for Catalysis. DOI:10.1016/j.jmb.2007.04.012. PMID:17493636.
  5. Zhang Y et al. (2004), Glycobiology, 14, 1295-1302. Roles of active site tryptophans in substrate binding and catalysis by  -1,3 galactosyltransferase. DOI:10.1093/glycob/cwh119. PMID:15229192.
  6. Boix E et al. (2002), J Biol Chem, 277, 28310-28318. Structural Basis of Ordered Binding of Donor and Acceptor Substrates to the Retaining Glycosyltransferase, alpha -1,3-Galactosyltransferase. DOI:10.1074/jbc.m202631200. PMID:12011052.
  7. Gastinel LN et al. (2001), EMBO J, 20, 638-649. Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases. DOI:10.1093/emboj/20.4.638. PMID:11179209.
  8. Boix E et al. (2001), J Biol Chem, 276, 48608-48614. Structure of UDP Complex of UDP-galactose:β-Galactoside-α-1,3-galactosyltransferase at 1.53-Å Resolution Reveals a Conformational Change in the Catalytically Important C Terminus. DOI:10.1074/jbc.m108828200. PMID:11592969.
  9. Zhang Y et al. (2001), J Biol Chem, 276, 11567-11574. Specificity and Mechanism of Metal Ion Activation in UDP-galactose:beta -Galactoside-alpha -1,3-galactosyltransferase. DOI:10.1074/jbc.m006530200. PMID:11133981.

Step 1. UDP is eliminated from the substrate to form an oxycarbenium ion transition state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Trp356(277)A electrostatic stabiliser, hydrogen bond donor
Glu317(238)A activator
Tyr314(235)A electrostatic stabiliser, hydrogen bond donor
Gln247(168)A hydrogen bond donor, electrostatic stabiliser
His280(201)A hydrogen bond donor, electrostatic stabiliser
Glu317(238)A electrostatic stabiliser
Arg365(286)A electrostatic stabiliser

Chemical Components

overall reactant used, overall product formed, proton transfer, ingold: unimolecular elimination by the conjugate base

Step 2. The activated polymer then adds to the oxycarbenium transition state to finish the reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln247(168)A electrostatic stabiliser
His280(201)A electrostatic stabiliser
Tyr314(235)A electrostatic stabiliser
Glu317(238)A electrostatic stabiliser
Trp356(277)A electrostatic stabiliser
Arg365(286)A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition
Download: Image, Marvin File

Step 1. UDP is eliminated from the substrate to form an oxycarbenium ion transition state.

Step 2. The activated polymer then adds to the oxycarbenium transition state to finish the reaction.

Products.

Introduction

The reaction proceeds via nucleophilic attack on the UDP galactose substrate by the catalytic residue Glu317 to form a covalent glycosyl-enzyme intermediate. This allows the galactose to be transferred to the second substrate through nucleophilic attack by the deprotonated sugar oxygen on the anomeric carbon. The double displacement thus results in retention of configuration (alpha). The mechanism whereby the sugar oxygen is deprotonated is unclear, but seems likely to involve direct transfer of the proton to the UDP leaving group.

Catalytic Residues Roles

UniProt PDB* (1vzx)
Glu317 Glu317(238)A Attacks the anomeric carbon of UDP galactose to form a covalent glycosyl-enzyme intermediate, allowing transfer of the galactose to the substrate. activator, covalently attached, nucleofuge, nucleophile
Trp314 Tyr314(235)A Stabilises the transition state in the galactose transfer hydrogen bond donor, electrostatic stabiliser
Trp356, Gln247, His280, Arg365 Trp356(277)A, Gln247(168)A, His280(201)A, Arg365(286)A Stabilises the transition state of the reaction. hydrogen bond donor, 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

bimolecular nucleophilic substitution, enzyme-substrate complex formation, overall reactant used, overall product formed, atom stereo change, enzyme-substrate complex cleavage, native state of enzyme regenerated

References

  1. Gastinel LN et al. (2001), EMBO J, 20, 638-649. Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases. DOI:10.1093/emboj/20.4.638. PMID:11179209.

Step 1. Glu317 displaces UDP from the anomeric carbon, forming an enzyme-substrate adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Trp356(277)A electrostatic stabiliser, hydrogen bond donor
Glu317(238)A activator
Tyr314(235)A electrostatic stabiliser, hydrogen bond donor
Gln247(168)A hydrogen bond donor, electrostatic stabiliser
His280(201)A hydrogen bond donor, electrostatic stabiliser
Arg365(286)A electrostatic stabiliser
Glu317(238)A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, overall reactant used, overall product formed, atom stereo change

Step 2. The beta-galactosyl acceptor substrate displaces Glu317. This second inversion of stereochemistry at the anomeric centre results in retention of the alpha configuration.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Trp356(277)A electrostatic stabiliser, hydrogen bond donor
Glu317(238)A activator, covalently attached
Tyr314(235)A electrostatic stabiliser, hydrogen bond donor
Gln247(168)A hydrogen bond donor, electrostatic stabiliser
His280(201)A hydrogen bond donor, electrostatic stabiliser
Arg365(286)A electrostatic stabiliser
Glu317(238)A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, atom stereo change, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu317 displaces UDP from the anomeric carbon, forming an enzyme-substrate adduct.

Step 2. The beta-galactosyl acceptor substrate displaces Glu317. This second inversion of stereochemistry at the anomeric centre results in retention of the alpha configuration.

Products.

Contributors

Sophie T. Williams, Peter Sarkies, Mei Leung, Gemma L. Holliday, Charity Hornby