Formate-dependent phosphoribosylglycinamide formyltransferase
The PurT encoded ribonucleotide transformylase is involved in the step of purine biosynthesis that is specific to bacteria, namely the formylation of GAR (glycinamide ribonucleotide). It displays sequence homology of around 20% to PurK, the enzyme which catalyses the equivalent reaction for the adenine biosynthetic pathway, but uses a different intermediate in the reaction, thus has a slightly different mechanism. Consequently, structural differences between the active sites of the two enzymes exist. However, the similarities are also important as both are members of the ATP grasp superfamily of enzymes that includes biotin carboxylase and sugar kinases amongst others, so analysis of the binding of ATP to this enzyme will give clues as to the overall evolutionary relationship between these enzymes.
Reference Protein and Structure
- Sequence
-
P33221
(2.1.2.-)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Escherichia coli K-12 (Bacteria)
- PDB
-
1ez1
- STRUCTURE OF ESCHERICHIA COLI PURT-ENCODED GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE COMPLEXED WITH MG, AMPPNP, AND GAR
(1.75 Å)
- Catalytic CATH Domains
-
3.30.1490.20
3.30.470.20
(see all for 1ez1)
- Cofactors
- Magnesium(2+) (3)
Enzyme Mechanism
Introduction
The reaction proceeds via initial nucleophilic attack by the formate substrate on ATP, forming a pentavalent phosphate transition state, stabilised by Mg2+, Ser 160 and Gly 162 at the active site. This releases ADP and the formyl phosphate intermediate that characterises this reaction mechanism as opposed to that catalysed by PurK for instance, where a carboxyphosphate intermediate is formed. This intermediate is then attacked by the nitrogen lone pair of GAR, with deprotonation achieved by Asp 286, which in turn is primed for its role by Arg 363 and Thr 287. This displaces the phosphate from the reaction intermediate to form the final product, formylated GAR.
Catalytic Residues Roles
| UniProt | PDB* (1ez1) | ||
| Asp286 | Asp286A | Deprotonates the amine of GAR to allow it to attack the formyl phosphate by nucleophilic attack to form the product. | hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor |
| Thr287 (main-N) | Thr287A (main-N) | Activates the Asp 286 towards its role as an acid base by forming contacts that maintain the correct protonation state of the residue. | hydrogen bond donor, electrostatic stabiliser |
| Ser160 | Ser160A | Forms contacts to the gamma phosphate of ATP, thus is able to stabilise the formyl phosphate intermediate through hydrogen bonding. | hydrogen bond donor, electrostatic stabiliser |
| Gly162 (main-N) | Gly162A (main-N) | Is able to form contacts to the gamma phosphate of ATP, thus stabilises the intermediate formyl phosphate that forms in the reaction. | hydrogen bond donor, electrostatic stabiliser |
Chemical Components
overall reactant used, bimolecular nucleophilic substitution, cofactor used, intermediate formation, intermediate collapse, overall product formed, proton transfer, dephosphorylation, inferred reaction step, native state of enzyme regeneratedReferences
- Thoden JB et al. (2000), Biochemistry, 39, 8791-8802. Molecular Structure ofEscherichia coliPurT-Encoded Glycinamide Ribonucleotide Transformylase†,‡. DOI:10.1021/bi000926j. PMID:10913290.
- Thoden JB et al. (2002), J Biol Chem, 277, 23898-23908. PurT-encoded Glycinamide Ribonucleotide Transformylase. ACCOMMODATION OF ADENOSINE NUCLEOTIDE ANALOGS WITHIN THE ACTIVE SITE. DOI:10.1074/jbc.m202251200. PMID:11953435.
Step 1. Formate acts as a nucleophile and attacks ATP, breaking the beta-gamma phosphate bond.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Thr287A (main-N) | electrostatic stabiliser |
| Ser160A | electrostatic stabiliser |
| Gly162A (main-N) | electrostatic stabiliser |
| Glu267A | metal ligand |
| Glu279A | metal ligand |
| Glu267A | electrostatic stabiliser |
| Glu279A | electrostatic stabiliser |
| Asp286A | hydrogen bond donor, hydrogen bond acceptor |
| Thr287A (main-N) | hydrogen bond donor |
| Arg363A | electrostatic stabiliser, hydrogen bond donor |
Chemical Components
overall reactant used, ingold: bimolecular nucleophilic substitution, cofactor used, intermediate formation
Step 2. Asp286 acts as a base to deprotonate the nitrogen of GAR. This nitrogen then attacks the carbonyl carbon of the formyl phosphate intermediate to form the product.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gly162A (main-N) | electrostatic stabiliser, hydrogen bond donor |
| Ser160A | electrostatic stabiliser, hydrogen bond donor |
| Glu267A | metal ligand |
| Glu279A | metal ligand |
| Glu267A | electrostatic stabiliser |
| Glu279A | electrostatic stabiliser |
| Thr287A (main-N) | hydrogen bond donor |
| Arg363A | hydrogen bond donor |
| Asp286A | hydrogen bond acceptor, metal ligand, proton acceptor |
Chemical Components
ingold: bimolecular nucleophilic substitution, intermediate collapse, overall product formed, proton transfer, dephosphorylation
Step 3. In an inferred mechanism step Asp286 is deprotonated to regenerate the native state of the enzyme.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asp286A | proton donor |
Download: