PDBsum entry: 3gar

Purine biosynthesis

PDB id: 3gar

Contents

Protein chain

209 a.a. *

Ligands

PO4

Waters ×148

* Residue conservation analysis

PDB id:

3gar

Name:

Purine biosynthesis

Title:

A ph-dependent stablization of an active site loop observed from low and high ph crystal structures of mutant monomeric glycinamide ribonucleotide transformylase

Structure:

Glycinamide ribonucleotide transformylase. Chain: a. Synonym: gartfase. Engineered: yes. Mutation: yes

Source:

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

Resolution:

1.90Å R-factor: 0.215 R-free: 0.274

Authors:

Y.Su,M.M.Yamashita,S.E.Greasley,C.A.Mullen,J.H.Shim, P.A.Jennings,S.J.Benkovic,I.A.Wilson

Key ref:

Y.Su et al. (1998). A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A. J Mol Biol, 281, 485-499. PubMed id: 9698564 DOI: 10.1006/jmbi.1998.1931

Date:

13-May-98 Release date: 12-Aug-98

Protein chain

P08179  (PUR3_ECOLI) -  Phosphoribosylglycinamide formyltransferase

Seq: 212 a.a.

Struc: 209 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

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: 10.1006/jmbi.1998.1931

J Mol Biol 281:485-499 (1998)

PubMed id: 9698564

A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A.

Y.Su, M.M.Yamashita, S.E.Greasley, C.A.Mullen, J.H.Shim, P.A.Jennings, S.J.Benkovic, I.A.Wilson.

ABSTRACT

A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the pKa of His108 is in fact raised to 9. 2. The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.

Selected figure(s)

Figure 2.
Figure 2. Structure of a GarT- fase dimer at low pH (<6.9) and location of mutation E70A. The dimerization inter- face of GarTfase is formed through symmetrical pairing of the outer strand, b3, of the central seven-stranded b-sheet and helices a2 and a3. The structure shown was deter- mined by Klein et al. (1995), PDB code 1GAR. Residues 38 to 78, in pink, form the dimer interface. The side-chains of residues Glu70 and His73 con- tribute significantly to the dimer association and are dis- played in green and purple, respectively. A mutation at Glu70 to Ala70 would disrupt specific electrostatic inter- actions at the dimer interface but would be expected to maintain the overall secondary structure in helix a3.

Figure 3.
Figure 3. Stereo view of crystal contacts in (a) the pH 3.5 E70A GarTfase in P212121 and in (b) the pH 7.5 E70A in P6122. Regions 141.145 and 158.166 are in green and the faces normally used for dimerization are in cyan. Note the complete loss of the normal dimer interface in both crystal forms.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 281, 485-499) copyright 1998.

Figures were selected by an automated process.