3B5B

Crystal structure of the thymidylate synthase k48q

3B5B の概要

• エントリーDOI: 10.2210/pdb3b5b/pdb
• 分子名称: Thymidylate synthase, 2'-DEOXY-5-NITROURIDINE 5'-MONOPHOSPHATE, FORMIC ACID, ... (5 entities in total)
• 機能のキーワード: thymidylate synthase, 5-no2-dump, substrate anologue, methyltransferase, nucleotide biosynthesis, repressor, rna-binding, transferase, translation regulation
• 由来する生物種: Escherichia coli BL21
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 61829.63

構造登録者

Sotelo-Mundo, R.R.,Arreola, R.,Maley, F.,Montfort, W.R. (登録日: 2007-10-25, 公開日: 2007-12-25, 最終更新日: 2023-08-30)

主引用文献

Arvizu-Flores, A.A.,Sugich-Miranda, R.,Arreola, R.,Garcia-Orozco, K.D.,Velazquez-Contreras, E.F.,Montfort, W.R.,Maley, F.,Sotelo-Mundo, R.R.
Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant.
Int.J.Biochem.Cell Biol., 40:2206-2217, 2008

PubMed Abstract: Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

PubMed: 18403248
DOI: 10.1016/j.biocel.2008.02.025

実験手法

X-RAY DIFFRACTION (2.7 Å)