1FFL

CRYSTAL STRUCTURE OF THE APO-THYMIDYLATE SYNTHASE R166Q MUTANT

Summary for 1FFL

• Entry DOI: 10.2210/pdb1ffl/pdb
• Descriptor: THYMIDYLATE SYNTHASE (2 entities in total)
• Functional Keywords: transferase, methyltransferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm : P0A884
• Total number of polymer chains: 1
• Total formula weight: 30530.60

Authors

Sotelo-Mundo, R.R.,Changchien, L.,Maley, F.,Montfort, W.R. (deposition date: 2000-07-25, release date: 2000-07-28, Last modification date: 2024-10-30)

Primary citation

Sotelo-Mundo, R.R.,Changchien, L.,Maley, F.,Montfort, W.R.
Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity.
J.Biochem.Mol.Toxicol., 20:88-92, 2006

PubMed Abstract: Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 A resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety.

PubMed: 16615077
DOI: 10.1002/jbt.20122

Experimental method

X-RAY DIFFRACTION (2.94 Å)