PDBsum entry: 1jkx

Transferase

PDB id: 1jkx

Contents

Protein chains

209 a.a. *

Ligands

138 ×4

Waters ×607

* Residue conservation analysis

PDB id:

1jkx

Name:

Transferase

Title:

Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of gar transformylase

Structure:

Phosphoribosylglycinamide formyltransferase. Chain: a, b, c, d. Fragment: transferase. Synonym: glycinamide ribonucleotide transformylase. Gart. Gar transformylase. 5'-phosphoribosylglycinamide transformylase. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: purn. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.60Å R-factor: 0.221 R-free: 0.243

Authors:

S.E.Greasley,T.H.Marsilje,H.Cai,S.Baker,S.J.Benkovic, D.L.Boger,I.A.Wilson

Key ref:

S.E.Greasley et al. (2001). Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase. Biochemistry, 40, 13538-13547. PubMed id: 11695901 DOI: 10.1021/bi011482+

Date:

13-Jul-01 Release date: 30-Nov-01

Protein chains

P08179  (PUR3_ECOLI) -  Phosphoribosylglycinamide formyltransferase

Seq: 212 a.a.

Struc: 209 a.a.

Enzyme reactions

• Enzyme class: E.C.2.1.2.2 - Phosphoribosylglycinamide formyltransferase.
• Pathway: Purine Biosynthesis (early stages)
• Reaction: 10-formyltetrahydrofolate + N₁-(5-phospho-D-ribosyl)glycinamide = tetrahydrofolate + N₂-formyl-N₁-(5-phospho-D-ribosyl)glycinamide

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: biosynthetic process (3 terms)
• Biochemical function: transferase activity (4 terms)

reference

DOI no: 10.1021/bi011482+

Biochemistry 40:13538-13547 (2001)

PubMed id: 11695901

Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase.

S.E.Greasley, T.H.Marsilje, H.Cai, S.Baker, S.J.Benkovic, D.L.Boger, I.A.Wilson.

ABSTRACT

Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake. Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site. We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM). However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct. Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.