3-phosphoshikimate 1-carboxyvinyltransferase

 

Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) catalyses the sixth step in aromatic amino acid biosynthesis in bacteria, plants and some parasites. It forms EPSP from shikimate 3-phosphate and phospho-enolpyruvate in an addition/elimination reaction the proceeds through a tetrahedral intermediate. Because this biosynthetic pathway is absent in animals, the enzyme is a potential antibacterial target with knock-out strains being avirulent.

 

Reference Protein and Structure

Sequence
P0A6D3 UniProt (2.5.1.19) IPR006264 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1g6t - STRUCTURE OF EPSP SYNTHASE LIGANDED WITH SHIKIMATE-3-PHOSPHATE (1.6 Å) PDBe PDBsum 1g6t
Catalytic CATH Domains
3.65.10.10 CATHdb (see all for 1g6t)
Cofactors
Potassium(1+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.19)

3-phosphonatoshikimate(3-)
CHEBI:145989ChEBI
+
phosphonatoenolpyruvate
CHEBI:58702ChEBI
→
5-O-(1-carboxylatovinyl)-3-phosphonatoshikimate
CHEBI:57701ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: 5-enolpyruvylshikimate-3-phosphate synthase, EPSP synthase, 3-enol-pyruvoylshikimate-5-phosphate synthase,

Enzyme Mechanism

Introduction

An aspartate residue deprotonates the 5-OH of the shikimate 3-phosphate, activating the oxygen to attack at the C2 of phospho-enolpyruvate with concomitant protonation at C3 by Glu341. A tetrahedral intermediate is formed. Glu341 is then thought to act as a general base at C3, initiating the collapse of the intermediate, elimination of phosphate and the formation of enolpyruvylshikimate 3-phosphate.

The most recent work suggests a hybrid acid/base- electrostatic reaction, with catalytic residues optimised for acid/base and electrostatic roles. Glu341 is thought to act as both a general acid, protonating C3 in the addition step, and a general base, deprotonating C3 in the elimination step while also stabilising the cationic intermediate. This is achieved by the formation of an electrostatic 'sandwich' with Asp313.

Catalytic Residues Roles

UniProt PDB* (1g6t)
Asp313 Asp313A The carboxylic side group is thought to activate the 5-OH of shikimate 3-phosphate towards attack at the C2 of phospho-enolpyruvate by proton abstraction. It is also positioned to stabilise the cationic intermediate, forming an electrostatic 'sandwich' with the close proximity Glu341. proton shuttle (general acid/base), electrostatic stabiliser
Glu341 Glu341A Acts as a general acid/base throughout the reaction, first donating a proton to the C3 of the enolpyruvyl-phosphate in the elimination step, and then abstracting a proton from C3 in the elimination step. This has been shown by stereo-chemical analysis, mutagenesis and structure determination. The residue is also thought to drive the reaction by stabilising the cationic intermediate in an electrostatic sandwich, and so lower the reaction barrier. The position of the side chain is influenced by hydrogen bonds to Lys411 and His385 through the residue's backbone amide. proton shuttle (general acid/base), metal ligand, electrostatic stabiliser
His385, Lys411 His385A, Lys411A Implicated in aligning the side chain of Glu341 for catalytic activity. steric role
Asp49, Glu341, Asn94 Asp49A, Glu341A, Asn94A Forms the cation binding site. metal ligand
Arg386 Arg386A Forms a tight bidentate ion pair at the transition state with the reactant's C1 carboxylate group. Acts to aid in stabilising the transition state. transition state 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

References

  1. Eschenburg S et al. (2003), J Biol Chem, 278, 49215-49222. A New View of the Mechanisms of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) and 5-Enolpyruvylshikimate-3-phosphate Synthase (AroA) Derived from X-ray Structures of Their Tetrahedral Reaction Intermediate States. DOI:10.1074/jbc.m309741200. PMID:13129913.
  2. Light SH et al. (2016), Biochemistry, 55, 1239-1245. An Unusual Cation-Binding Site and Distinct Domain–Domain Interactions Distinguish Class II Enolpyruvylshikimate-3-phosphate Synthases. DOI:10.1021/acs.biochem.5b00553. PMID:26813771.
  3. Lou M et al. (2012), J Am Chem Soc, 134, 12958-12969. Transition State Analysis of Enolpyruvylshikimate 3-Phosphate (EPSP) Synthase (AroA)-Catalyzed EPSP Hydrolysis. DOI:10.1021/ja304339h. PMID:22765279.
  4. Lou M et al. (2012), J Am Chem Soc, 134, 12947-12957. Transition State Analysis of Acid-Catalyzed Hydrolysis of an Enol Ether, Enolpyruvylshikimate 3-Phosphate (EPSP). DOI:10.1021/ja3043382. PMID:22765168.
  5. Berti PJ et al. (2009), Biochemistry, 48, 3699-3707. Catalytic Residues and an Electrostatic Sandwich That Promote Enolpyruvyl Shikimate 3-Phosphate Synthase (AroA) Catalysis†. DOI:10.1021/bi802251s. PMID:19271774.
  6. de Souza AX et al. (2008), Bioorg Chem, 36, 113-120. 5-Enolpyruvylshikimate-3-phosphate synthase: Determination of the protonation state of active site residues by the semiempirical method. DOI:10.1016/j.bioorg.2007.12.007. PMID:18325563.
  7. Park H et al. (2004), Mol Microbiol, 51, 963-971. Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state. DOI:10.1046/j.1365-2958.2003.03885.x.
  8. Schönbrunn E et al. (2001), Proc Natl Acad Sci U S A, 98, 1376-1380. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. DOI:10.1073/pnas.98.4.1376. PMID:11171958.
  9. Du W et al. (2000), Eur J Biochem, 267, 222-227. Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations. PMID:10601870.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu341A electrostatic stabiliser, proton shuttle (general acid/base)
Asp313A electrostatic stabiliser, proton shuttle (general acid/base)
His385A steric role
Lys411A steric role
Asp49A metal ligand
Asn94A metal ligand
Glu341A metal ligand
Arg386A transition state stabiliser

Chemical Components

Step 1.

Contributors

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