Pantetheine-phosphate adenylyltransferase

 

Phosphopantetheine adenylyltransferase (PPAT), isolated from Escherichia coli, catalyses the magnesium-dependent adenylyl transfer from ATP to 4'-phosphopantetheine (Ppant or PhP) to form dephospho-CoA (dPCoA). This reaction is the penultimate step in the synthesis of CoA. PPAT belongs to the nucleotidyltransferase alpha/beta phosphodiesterase superfamily, whose members catalyse the transfer of a nucleotide monophosphate to a substrate by stabilising the transition state of the reaction.

PPAT is a hexamer consisting of two trimers. While each subunit possesses an active site, it appears that only the subunits of one trimer will catalyse the reaction at a given time. The reaction proceeds via a random bi-bi mechanism in that the order of the binding of ATP and Ppant is not fixed, nor is the release of dPCoA and pyrophosphate. CoA can regulate the activity of PPAT by binding to the PPAT.PPi complex, thus preventing the binding of a new Ppant substrate molecule.

 

Reference Protein and Structure

Sequence
P0A6I6 UniProt (2.7.7.3) IPR001980 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1b6t - PHOSPHOPANTETHEINE ADENYLYLTRANSFERASE IN COMPLEX WITH 3'-DEPHOSPHO-COA FROM ESCHERICHIA COLI (1.8 Å) PDBe PDBsum 1b6t
Catalytic CATH Domains
3.40.50.620 CATHdb (see all for 1b6t)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.7.7.3)

D-pantetheine 4'-phosphate(2-)
CHEBI:61723ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
hydron
CHEBI:15378ChEBI
→
3'-dephospho-CoA(2-)
CHEBI:57328ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: 3'-dephospho-CoA pyrophosphorylase, Dephospho-CoA pyrophosphorylase, Dephospho-coenzyme A pyrophosphorylase, Pantetheine phosphate adenylyltransferase, PPAT, Dephospho-CoA diphosphorylase, Phosphopantetheine adenylyltransferase,

Enzyme Mechanism

Introduction

One of the 4'-phosphate oxygen's of Ppant undergoes nucleophilic attack on the alpha-phosphate group of ATP with pyrophosphate as the leaving group. The pentacovalent transition state is stabilised by His18 while the leaving group is stabilised by Arg91, Ser129 and the magnesium ion.

Catalytic Residues Roles

UniProt PDB* (1b6t)
His18 His18A Stabilises the pentacovalent transition state by forming a hydrogen bond to the non-ester oxygens of P(alpha). hydrogen bond donor, electrostatic stabiliser
Ser129 (main-N), Lys42, Arg91 Ser129A (main-N), Lys42A, Arg91A Stabilises the beta-phosphate, making pyrophosphate a better leaving group. 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, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Izard T et al. (1999), EMBO J, 18, 2021-2030. The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity. DOI:10.1093/emboj/18.8.2021. PMID:10205156.
  2. Izard T (2002), J Mol Biol, 315, 487-495. The crystal structures of phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme’s catalytic mechanism. DOI:10.1006/jmbi.2001.5272. PMID:11812124.

Step 1. The ATP adopts a strained conformation, as seen in the active sites of aminoacyl-tRNA synthetase enzymes. This raises the energy of the ground state towards that of the transition state, lowering the overall activation energy required for the reaction [PMID:10205156]. The phosphate group of pantetheine 4'-phosphate initiates a nucleophilic attack on the alpha phosphate group of ATP in a substitution reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg91A attractive charge-charge interaction, electrostatic stabiliser
Lys42A attractive charge-charge interaction, electrostatic stabiliser
His18A hydrogen bond donor, electrostatic stabiliser
Ser129A (main-N) hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The ATP adopts a strained conformation, as seen in the active sites of aminoacyl-tRNA synthetase enzymes. This raises the energy of the ground state towards that of the transition state, lowering the overall activation energy required for the reaction [PMID:10205156]. The phosphate group of pantetheine 4'-phosphate initiates a nucleophilic attack on the alpha phosphate group of ATP in a substitution reaction.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday