PMID:16615077

Citation	Sotelo-Mundo, RR, Changchien, L, Maley, F and Montfort, WR (2006) Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity. J. Biochem. Mol. Toxicol. 20:88-92
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.
Links	PubMed Online version:10.1002/jbt.20122
Keywords	Amino Acid Substitution; Arginine/metabolism; Binding Sites; Catalysis; Crystallization; Crystallography, X-Ray; Deoxyuracil Nucleotides/metabolism; Dimerization; Escherichia coli/enzymology; Folic Acid/analogs & derivatives; Folic Acid/metabolism; Folic Acid Antagonists/metabolism; Hydrogen Bonding; Kinetics; Ligands; Models, Molecular; Molecular Structure; Protein Binding; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Thymidine Monophosphate/metabolism; Thymidylate Synthase/chemistry; Thymidylate Synthase/genetics; Thymidylate Synthase/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:A0A0K2D0F3		GO:0004799: thymidylate synthase activity	ECO:0000250:	UniProtKB:A0A023Z1P7	F		"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." meaning that this protein has a TS sight and if this site was mutated from a R to a Q at the 166 peptide, the site was inactive. This has a similar sequence from the TS in E.coli.	complete CACAO 11431
ECOLX:A0A023Z1P7		GO:2000966: regulation of cell wall polysaccharide catabolic process	ECO:0000250:	UniProtKB:A0A0G4BQJ3	P		MltA is a lytic transglycosylase of Gram-negative bacteria that cleaves the beta-1,4 glycosidic linkages between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan. We have determined the crystal structures of MltA from Neisseria gonorrhoeae and Escherichia coli (NgMltA and EcMltA), which have only 21.5% sequence identity but have 2 main domains separated by a deep grove pointing that they are both MltA proteins.	complete CACAO 11434

References

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