Alpha,alpha-trehalose-phosphate synthase (UDP-forming)

 

Escherichia coli trehalose-6-phosphate synthase is part of the glycosyl transferase family 20, the retaining glycosyl transferases. It is able to catalyse the condensation between Glucose-6-phosphate and UDP-glucose to form trehalose-6-phosphate, an important metabolite for many bacteria and plants. Interest in the enzyme stems both from the study of the unusual reaction mechanism and the fact that it plays a key role in bacterial cell wall synthesis in M. tuberculosis, so is a possible target for antibiotics. It displays sequence and structural identity with the glycogen phosphorylases in particular, suggesting a common mechanism and evolutionary origin. Also found to be similar is the pseudo-glycosyltransferase VldE involved in validoxylamine A 7'-phosphate synthesis.

 

Reference Protein and Structure

Sequence
P31677 UniProt (2.4.1.15) IPR001830 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1uqt - Trehalose-6-phosphate from E. coli bound with UDP-2-fluoro glucose. (2.0 Å) PDBe PDBsum 1uqt
Catalytic CATH Domains
3.40.50.2000 CATHdb (see all for 1uqt)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.15)

D-glucopyranose 6-phosphate(2-)
CHEBI:61548ChEBI
+
UDP-alpha-D-glucose(2-)
CHEBI:58885ChEBI
→
alpha,alpha-trehalose 6-phosphate(2-)
CHEBI:58429ChEBI
+
UDP(3-)
CHEBI:58223ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Alpha,alpha-trehalose phosphate synthase (UDP-forming), UDP-glucose--glucose-phosphate glucosyltransferase, Phosphotrehalose-uridine diphosphate transglucosylase, Transglucosylase, Trehalose 6-phosphate synthase, Trehalose 6-phosphate synthetase, Trehalose phosphate synthase, Trehalose phosphate synthetase, Trehalose phosphate-uridine diphosphate glucosyltransferase, Trehalose-P synthetase, Trehalosephosphate-UDP glucosyltransferase, Uridine diphosphoglucose phosphate glucosyltransferase, UDP-glucose:D-glucose-6-phosphate 1-alpha-D-glucosyltransferase,

Enzyme Mechanism

Introduction

The reaction mechanism proceeds via an SNi type, meaning internal return whereby nucleophilic attack occurs on the same side as the leaving group departs. The ring oxygen of the UDP glucose donates electron density to C1 to allow the UDP to leave the molecule. This forms a shortly lived oxocarbenium ion stabilised by His 154 and Asp 361. Nucleophilic attack from the glucose-6-phosphate OH group on the anomeric carbon of glucose ensures that the configuration is retained to give the disaccharide product. UDP phosphate forming a hydrogen bond to the OH 1 on the acceptor molecule stabilises the negative charges developing on pyrophosphate, promoting UDP as a leaving group and also positions glucose-6-phosphate for nucleophilic attack. The activation of the OH towards nucleophilic attack is accomplished by the UDP moiety acting as a general base to remove a proton.

Catalytic Residues Roles

UniProt PDB* (1uqt)
His155 (main-C) His154(155)A (main-C) Carbonyl oxygen lone pair contributes electron density to the positively charged ring oxygen to stabilise the oxonium ion transition state that forms as Glucose-6-phosphate attacks UDP glucose. electrostatic stabiliser
Asp362 Asp361(362)A The oxonium ion's different structure relative to the substrate pushes its 3C OH group into alignment with the side chain carbonyl of Asp 361, thus allowing a hydrogen bond to form stabilising the transition state for the reaction. 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

heterolysis, overall reactant used, intermediate formation, elimination (not covered by the Ingold mechanisms), proton transfer, overall product formed, bimolecular nucleophilic substitution

References

  1. Ardèvol A et al. (2011), Angew Chem Weinheim Bergstr Ger, 123, 11089-11093. The Molecular Mechanism of Enzymatic Glycosyl Transfer with Retention of Configuration: Evidence for a Short-Lived Oxocarbenium-Like Species. DOI:10.1002/ange.201104623.
  2. Ardèvol A et al. (2016), Biochem Soc Trans, 44, 51-60. The reaction mechanism of retaining glycosyltransferases. DOI:10.1042/BST20150177. PMID:26862188.
  3. Cavalier MC et al. (2012), PLoS One, 7, e44934-. Mechanistic insights into validoxylamine A 7'-phosphate synthesis by VldE using the structure of the entire product complex. DOI:10.1371/journal.pone.0044934. PMID:23028689.
  4. Gibson RP et al. (2004), J Biol Chem, 279, 1950-1955. The donor subsite of trehalose-6-phosphate synthase: binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 A resolution. DOI:10.1074/jbc.M307643200. PMID:14570926.
  5. Gibson RP et al. (2002), Chem Biol, 9, 1337-1346. Insights into Trehalose Synthesis Provided by the Structure of the Retaining Glucosyltransferase OtsA. DOI:10.1016/s1074-5521(02)00292-2. PMID:12498887.

Step 1. Cleavage of glycosidic bond between C1 and O while hydrogen bonds form between the OH on acceptor and O of phosphate.

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Catalytic Residues Roles

Residue Roles
Asp361(362)A electrostatic stabiliser
His154(155)A (main-C) electrostatic stabiliser

Chemical Components

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

Step 2. Oxocarbenium ion intermediate generated is very short-lived. Concomitant Proton transfer and nucleophilic attack of O1 on glucose-6-phosphate on anomeric carbon on glucose intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His154(155)A (main-C) electrostatic stabiliser
Asp361(362)A electrostatic stabiliser

Chemical Components

proton transfer, overall product formed, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. Cleavage of glycosidic bond between C1 and O while hydrogen bonds form between the OH on acceptor and O of phosphate.

Step 2. Oxocarbenium ion intermediate generated is very short-lived. Concomitant Proton transfer and nucleophilic attack of O1 on glucose-6-phosphate on anomeric carbon on glucose intermediate.

Products.

Contributors

Hannah Gilbert, Peter Sarkies, Gemma L. Holliday, Morwenna Hall