PDBsum entry 3b5b

Transferase

PDB id: 3b5b

Contents

Protein chains

264 a.a. *

Ligands

NDU

FMT ×2

NDN

Waters ×52

* Residue conservation analysis

PDB id:

3b5b

Name:

Transferase

Title:

Crystal structure of the thymidylate synthase k48q

Structure:

Thymidylate synthase. Chain: a, b. Synonym: ts, tsase. Engineered: yes. Mutation: yes

Source:

Escherichia coli bl21. Organism_taxid: 511693. Strain: bl21. Gene: thya. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

2.70Å R-factor: 0.193 R-free: 0.234

Authors:

R.R.Sotelo-Mundo,R.Arreola,F.Maley,W.R.Montfort

Key ref:

A.A.Arvizu-Flores et al. (2008). 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. PubMed id: 18403248 DOI: 10.1016/j.biocel.2008.02.025

Date:

25-Oct-07 Release date: 25-Dec-07

Protein chains

P0A884  (TYSY_ECOLI) -  Thymidylate synthase from Escherichia coli (strain K12)

Seq: 264 a.a.

Struc: 264 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.1.1.45 - thymidylate synthase.
• Pathway: Folate Coenzymes
• Reaction: dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-dihydrofolate + dTMP

dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate (Bound ligand (Het Group name = NDU) matches with 86.96% similarity) = 7,8-dihydrofolate + dTMP

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.biocel.2008.02.025

Int J Biochem Cell Biol 40:2206-2217 (2008)

PubMed id: 18403248

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant.

A.A.Arvizu-Flores, R.Sugich-Miranda, R.Arreola, K.D.Garcia-Orozco, E.F.Velazquez-Contreras, W.R.Montfort, F.Maley, R.R.Sotelo-Mundo.

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.