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Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity
Journal of biochemical and molecular toxicology, 2006-01, Vol.20 (2), p.88-92
Sotelo-Mundo, Rogerio R.
Changchien, Liming
Maley, Frank
Montfort, William R.
2006
Details
Autor(en) / Beteiligte
Sotelo-Mundo, Rogerio R.
Changchien, Liming
Maley, Frank
Montfort, William R.
Titel
Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity
Ist Teil von
Journal of biochemical and molecular toxicology, 2006-01, Vol.20 (2), p.88-92
Ort / Verlag
Hoboken: Wiley Subscription Services, Inc., A Wiley Company
Erscheinungsjahr
2006
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
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 Å 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. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:88–92, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20122
Sprache
Englisch
Identifikatoren
ISSN: 1095-6670
eISSN: 1099-0461
DOI: 10.1002/jbt.20122
Titel-ID: cdi_proquest_miscellaneous_20861104
Format
–
Schlagworte
Amino Acid Substitution
,
Antifolate
,
Arginine - metabolism
,
Binding Sites
,
Catalysis
,
Crystallization
,
Crystallography, X-Ray
,
Deoxyuracil Nucleotides - metabolism
,
Dimerization
,
Enzyme Mechanism
,
Escherichia coli
,
Escherichia coli - enzymology
,
Folic Acid - analogs & derivatives
,
Folic Acid - metabolism
,
Folic Acid Antagonists - metabolism
,
Hydrogen Bonding
,
Kinetics
,
Ligand Binding
,
Ligands
,
Models, Molecular
,
Molecular Structure
,
Mutant
,
Protein Binding
,
Protein Conformation
,
Recombinant Proteins - chemistry
,
Recombinant Proteins - isolation & purification
,
Recombinant Proteins - metabolism
,
Thymidine Monophosphate - metabolism
,
Thymidylate Synthase
,
Thymidylate Synthase - chemistry
,
Thymidylate Synthase - genetics
,
Thymidylate Synthase - metabolism
,
X-ray Crystallography
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