PDBsum entry 5tmp

Transferase

PDB id: 5tmp

Contents

Protein chain

210 a.a. *

Ligands

Z5A

Waters ×72

* Residue conservation analysis

PDB id:

5tmp

Name:

Transferase

Title:

Complex of e. Coli thymidylate kinase with the bisubstrate inhibitor aztp5a

Structure:

Protein (thymidylate kinase). Chain: a. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PQS)

Resolution:

1.98Å R-factor: 0.204 R-free: 0.253

Authors:

A.Lavie,N.Ostermann,I.Schlichting

Key ref:

A.Lavie et al. (1998). Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase. Proc Natl Acad Sci U S A, 95, 14045-14050. PubMed id: 9826650 DOI: 10.1073/pnas.95.24.14045

Date:

01-Sep-98 Release date: 25-Nov-98

Protein chain

P0A720  (KTHY_ECOLI) -  Thymidylate kinase from Escherichia coli (strain K12)

Seq: 213 a.a.

Struc: 210 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.7.4.9 - dTMP kinase.
• Reaction: dTMP + ATP = dTDP + ADP

dTMP (Bound ligand (Het Group name = Z5A) matches with 54.39% similarity) + ATP = dTDP + ADP

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

reference

DOI no: 10.1073/pnas.95.24.14045

Proc Natl Acad Sci U S A 95:14045-14050 (1998)

PubMed id: 9826650

Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.

A.Lavie, N.Ostermann, R.Brundiers, R.S.Goody, J.Reinstein, M.Konrad, I.Schlichting.

ABSTRACT

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5'-adenosyl)-P5-(5'-thymidyl)pentaphosphate and pentaphosphate have been solved to 2.0-A and 2.2-A resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3'-azido-3'-deoxythymidine 5'-monophosphate (AZT-MP) at a 200-fold reduced turnover number (kcat) in comparison to the physiological substrate dTMP, reduction of kcat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3'-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3'-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3' substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.

Selected figure(s)

Figure 1.
Fig. 1. Interactions of the bisubstrate inhibitor with TmpK (a). Distance map of TP[5]A bound to TmpK[coli]. P loop residues are marked with an asterisk. (b-d) Stereoviews. Overlay of the TmpK[coli]-TP[5]A complex model (pink) with the TmpK[yeast]-TP[5]A model (green) (b and c) or the TmpK[coli]-AZTP[5]A (blue) (d). (b) Interactions of the 3'-hydroxyl of the thymidine deoxyribose. In TmpK[yeast], a bidentate interaction between the P loop aspartic acid and the sugar hydroxyl is observed. The binding of AZT-MP causes the P loop to move, thus displacing the catalytic P loop arginine. In contrast, in TmpK[coli], the interaction between Glu-12 and the 3'-hydroxyl is side-on, and the bulkier azido group does not induce a significant movement of the P loop. (c) Similar phosphate-arginine interactions made in TmpK[yeast] by Arg-15 and in TmpK[coli] by Arg-153. Displayed are the P loop and a part of the Lid region. The structures were overlaid according to the position of the bisubstrate inhibitor. (d) In the TmpK[coli]-TP[5]A and the TmpK[coli]-AZTP[5]A complex structures, the thymine base is at an identical position, but the deoxyribose moiety has undergone a rigid-body rotation caused by the azido group in the AZT-P[5]A complex. In addition, Glu-12 has rotated slightly to provide more room for the azido group. The rotation of the deoxyribose induces a similar rotation of the Glu-160 side chain. As Glu-12 makes close interactions with Asp-157, the latter carboxylic acid also rotates slightly. b-d were generated by using BOBSCRIPT (29, 30) and RASTER 3D (31).

Figure 2.
Fig. 2. Structure-based sequence alignment of the P loop and Lid regions. Lysine and arginine residues that make phosphate interactions are underlined doubly and those that make a stacking interaction with the adenine base are underlined singly. TmpKs are unique in having a carboxylic acid situated at the tip of the P loop (Glu-12 in TmpK[coli]). In type I TmpKs (e.g., human and yeast), the following residue is an arginine that has been shown to be catalytically important for the yeast enzyme. Type II TmpKs (e.g., E. coli) lack this arginine, having instead a number of basic residues in their Lid region: for TmpK[coli], Arg-153 presumably fulfills a catalytically role analogous to that of Arg-15 in yeast. Although the last Lid arginine in TmpKs (Arg-158 in TmpK[coli]) aligns well with catalytic arginines from pig adenylate kinase (AK[pig]) and Dictyostelium uridylate kinase (UmpK[dicty]), it points away from the active site, thus having no obvious catalytic role. The eukaryotic TmpKs have no catalytic residues in the Lid region.