Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

 

N-acetylglucosaminyltransferase I (GnT I) is an inverting glycosyl transferase from Oryctolagus cuniculus catalyses the attachment of N-acetyl glucosamine (GlcNAc) onto an oligomannose core, from UPD-N-acetyl glucosamine. It plays a critical role in mammalian development, involved in the transformation of oligomannose attached structures to complex N-glycans.

 

Reference Protein and Structure

Sequence
P27115 UniProt (2.4.1.101) IPR004139 (Sequence Homologues) (PDB Homologues)
Biological species
Oryctolagus cuniculus (rabbit) Uniprot
PDB
1foa - CRYSTAL STRUCTURE OF N-ACETYLGLUCOSAMINYLTRANSFERASE I (1.8 Å) PDBe PDBsum 1foa
Catalytic CATH Domains
3.90.550.10 CATHdb (see all for 1foa)
Cofactors
Manganese(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.101)

alpha-D-mannosyl-(1->3)-beta-D-mannosyl group
CHEBI:32591ChEBI
+
UDP-N-acetyl-alpha-D-glucosamine(2-)
CHEBI:57705ChEBI
→
N-acetyl-beta-D-glucosaminyl-(1->2)-alpha-D-mannosyl-(1->3)-beta-D-mannosyl group
CHEBI:32618ChEBI
+
UDP(3-)
CHEBI:58223ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: N-acetylglucosaminyltransferase I, N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase I, Alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, UDP-N-acetylglucosaminyl:alpha-1,3-D-mannoside-beta-1,2-N-acetylglucosaminyltransferase I, UDP-N-acetylglucosaminyl:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I, Uridine diphosphoacetylglucosamine-alpha-1,3-mannosylglycoprotein beta-1,2-N-acetylglucosaminyltransferase, GnTI, GlcNAc-T I, UDP-N-acetyl-D-glucosamine:3-(alpha-D-mannosyl)-beta-D-mannosyl-glycoprotein 2-beta-N-acetyl-D-glucosaminyltransferase,

Enzyme Mechanism

Introduction

This reaction occurs in an SN2 concerted mechanism, whereby induced fit interactions help destabilise the ground state promoting catalysis. Asp 291 acts as a general base to deprotonate a hydroxyl group of the oligomannose core sugar. The deprotonated oxygen atom then acts as a nucleophile and attacks C1 of UPD-N-acetyl glucosamine. The transition state is oxocarbenium ion-like. The C1 - UPD bond is broken, leaving the N-acetyl glucosamine bonded to the oligomannose core. Mn2+ stabilises the build up of negative charge on the beta-phosphate of the UPD. It is thought that Asp 291 then acts as an acid to protonate the UPD leaving group.

Catalytic Residues Roles

UniProt PDB* (1foa)
Asp213 Asp213(114)A Coordinates to Mn2+ ion, which is involved in stabilising the transition state. metal ligand
Asp291 Asp291(192)A Asp 291 acts as a general base by accepting a proton from a hydroxyl group on the oligamannose core. It must then donate that proton back to the leaving group UDP phosphate ion. 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

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

References

  1. Tvaroska I et al. (2003), Glycobiology, 13, 559-566. Catalytic mechanism of the inverting N-acetylglucosaminyltransferase I: DFT quantum mechanical model of the reaction pathway and determination of the transition state structure. DOI:10.1093/glycob/cwg067. PMID:12672701.
  2. Tvaroška I (2015), Carbohydr Res, 403, 38-47. Atomistic insight into the catalytic mechanism of glycosyltransferases by combined quantum mechanics/molecular mechanics (QM/MM) methods. DOI:10.1016/j.carres.2014.06.017. PMID:25060837.
  3. Unligil UM et al. (2000), EMBO J, 19, 5269-5280. X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. DOI:10.1093/emboj/19.20.5269. PMID:11032794.

Step 1. Asp291 deprotonates the C2- OH on the mannose sugar, activating it for nucleophilic attack on C1 of UDP-GlcNAc.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp213(114)A metal ligand
Asp291(192)A activator
Asp291(192)A proton acceptor

Chemical Components

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

Step 2. Asp291 is deprotonated by the leaving UDP group phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp213(114)A metal ligand
Asp291(192)A proton donor

Chemical Components

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

Step 1. Asp291 deprotonates the C2- OH on the mannose sugar, activating it for nucleophilic attack on C1 of UDP-GlcNAc.

Step 2. Asp291 is deprotonated by the leaving UDP group phosphate.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Morwenna Hall