2VF0

CRYSTAL STRUCTURE OF THE THYMIDYLATE SYNTHASE K48Q COMPLEXED WITH 5NO2DUMP AND BW1843U89

Summary for 2VF0

• Entry DOI: 10.2210/pdb2vf0/pdb
• Related: 1AIQ 1AJM 1AN5 1AOB 1AXW 1BDU 1BID 1BJG 1BQ1 1BQ2 1DDU 1DNA 1EV5 1EV8 1EVF 1EVG 1F4B 1F4C 1F4D 1F4E 1F4F 1F4G 1FFL 1FWM 1JG0 1JTQ 1JTU 1JUT 1KCE 1KZI 1KZJ 1NCE 1QQQ 1SYN 1TDU 1TJS 1TLC 1TLS 1TRG 1TSD 1TSN 1TYS 1ZPR 2A9W 2BBQ 2FTN 2FTO 2FTQ 2G8M 2G8O 2G8X 2KCE 2TSC 2VET 3B5B 3TMS
• Descriptor: THYMIDYLATE SYNTHASE, 2'-DEOXY-5-NITROURIDINE 5'-MONOPHOSPHATE, S)-2-(5(((1,2-DIHYDRO-3-METHYL-1-OXOBENZO(F)QUINAZOLIN-9-YL)METHYL)AMINO)1-OXO-2-ISOINDOLINYL)GLUTARIC ACID, ... (5 entities in total)
• Functional Keywords: nucleotide biosynthesis, substrate anologue, antifolate binding, rna-binding, transferase, methyltransferase, repressor, bw1843u89, cytoplasm, 5-no2-dump, thymidylate synthase, translation regulation
• Biological source: ESCHERICHIA COLI
• Cellular location: Cytoplasm : P0A884
• Total number of polymer chains: 2
• Total formula weight: 62924.69

Authors

Sotelo-Mundo, R.R.,Arreola, R.,Maley, F.,Montfort, W.R. (deposition date: 2007-10-27, release date: 2007-12-04, Last modification date: 2024-11-06)

Primary citation

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-, 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

Experimental method

X-RAY DIFFRACTION (3 Å)