Glycinamide ribonucleotide transformylase

 

Glycinamide ribonucleotide transformylase (GRT) catalyses the first two steps of purine biosynthesis: the transfer of formyl from 10-formyltetrahydrofolate to the amino group of glycinamide ribonucleotidem, forming glycinamide ribonucleotide and tetrahydrofolate.

The enzyme’s role in DNA nucleotide biosynthesis makes it an interesting target for anti-neoplastic agents and for developing novel antifolate drugs to be used in cancer chemotherapy. Due to its biological and pharmacological significance, it has been the subject of intensive studies.

 

Reference Protein and Structure

Sequence
P08179 UniProt (2.1.2.2) IPR004607 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1grc - CRYSTAL STRUCTURE OF GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE FROM ESCHERICHIA COLI AT 3.0 ANGSTROMS RESOLUTION: A TARGET ENZYME FOR CHEMOTHERAPY (3.0 Å) PDBe PDBsum 1grc
Catalytic CATH Domains
3.40.50.170 CATHdb (see all for 1grc)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:2.1.2.2)

10-formyltetrahydrofolate(2-)
CHEBI:57454ChEBI
+
N(1)-(5-phospho-D-ribosyl)glycinamide(1-)
CHEBI:58457ChEBI
→
(6S)-5,6,7,8-tetrahydrofolate(2-)
CHEBI:57453ChEBI
+
N(2)-formyl-N(1)-(5-phospho-D-ribosyl)glycinamide(2-)
CHEBI:58426ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 2-amino-N-ribosylacetamide 5'-phosphate transformylase, 5,10-methenyltetrahydrofolate:2-amino-N-ribosylacetamide ribonucleotide transformylase, GAR TFase, GAR formyltransferase, GAR transformylase, Glycinamide ribonucleotide transformylase, GART, 5'-phosphoribosylglycinamide transformylase,

Enzyme Mechanism

Introduction

Folate binding induces active site rearrangements involving Asp144 and His108. Asp144 forms a salt bridge to the imidazolium of His108 and the formyl group is positioned to form hydrogen bonds to Asn106 and the protonated imidazolium group of His108. Asn106 may fine-tune the location of the formyl group.

A low-dielectric active site environment favours the free base form of the amino group of GAR, poised to attack the activated formyl group to form presumably a tetrahedral intermediate in the transfer process. A proton is switched from GAR to the N10 of folate mediated by a catalytic water molecule, followed by breakdown of the tetrahedral intermediate and product release. The positioning of the catalytic water molecule may also be assisted by Asp144.

Catalytic Residues Roles

UniProt PDB* (1grc)
Ser135 Ser135A Helps stabilise the reactive intermediates and transition states formed during the course of the reaction. hydrogen bond donor, steric role, electrostatic stabiliser
Asp144 Asp144A Forms a salt bridge with His108. Essential as maintains the correct protonation state of the His108. Asp144 activates a tightly bound water molecule via hydrogen bonding. It is also possible that it acts as a general acid/base (again, via the conserved water molecule) donating a proton to the N10 atom of THF, enabling the formyl group to leave. attractive charge-charge interaction, hydrogen bond acceptor, electrostatic stabiliser, increase electrophilicity
His108, Asn106 His108A, Asn106A The Asn and His residues are both presumed to stabilise the oxyanion group of the GAR-formyl-THF intermediate, supported by the observed conformations of these residues in the structure of the enzyme bound to U89, believed to be a transition state analogue. In addition, the His residue may well rotate substantially to contact Ser135, leaving Asn106 to stabilise the anion. attractive charge-charge interaction, hydrogen bond donor, activator, electrostatic stabiliser, increase electrophilicity
*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 addition, overall reactant used, intermediate formation, elimination (not covered by the Ingold mechanisms), overall product formed

References

  1. Shim JH et al. (1999), Biochemistry, 38, 10024-10031. Catalytic Mechanism ofEscherichia coliGlycinamide Ribonucleotide Transformylase Probed by Site-Directed Mutagenesis and pH-Dependent Studies†. DOI:10.1021/bi9904609. PMID:10433709.
  2. Warren MS et al. (1996), Biochemistry, 35, 8855-8862. A Rapid Screen of Active Site Mutants in Glycinamide Ribonucleotide Transformylase†. DOI:10.1021/bi9528715. PMID:8688421.
  3. Almassy RJ et al. (1992), Proc Natl Acad Sci U S A, 89, 6114-6118. Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase. DOI:10.1073/pnas.89.13.6114. PMID:1631098.

Step 1. The free base form of glycinamide ribonucleotide attacks the activated formyl group to form an anionic tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His108A activator, hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity
Asn106A electrostatic stabiliser, increase electrophilicity
Asp144A hydrogen bond acceptor, attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity
Ser135A hydrogen bond donor, steric role, electrostatic stabiliser
Asn106A hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 2. The tetrahedral intermediate collapses, to produce the formylated phosphor-ribosyl glycinamide with concomitant reprotonation of the tetrafolate through a conserved water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His108A activator, hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity
Asn106A electrostatic stabiliser, increase electrophilicity
Asp144A hydrogen bond acceptor, attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity
Ser135A hydrogen bond donor, steric role
Asn106A hydrogen bond donor

Chemical Components

elimination (not covered by the Ingold mechanisms), overall reactant used, overall product formed
Download: Image, Marvin File

Step 1. The free base form of glycinamide ribonucleotide attacks the activated formyl group to form an anionic tetrahedral intermediate.

Step 2. The tetrahedral intermediate collapses, to produce the formylated phosphor-ribosyl glycinamide with concomitant reprotonation of the tetrafolate through a conserved water molecule.

Products.

Contributors

Sophie T. Williams, Nozomi Nagano, Anna Waters, Craig Porter, Gemma L. Holliday