PDBsum entry 1e9d

Transferase

PDB id: 1e9d

Contents

Protein chain

210 a.a. *

Ligands

ATM

ADP

Metals

_MG ×2

Waters ×199

* Residue conservation analysis

PDB id:

1e9d

Name:

Transferase

Title:

Mutant human thymidylate kinase (f105y) complexed with aztmp and adp

Structure:

Thymidylate kinase. Chain: a. Synonym: dtmp kinase. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Homo-Dimer (from PDB file)

Resolution:

1.70Å R-factor: 0.202 R-free: 0.257

Authors:

N.Ostermann,A.Lavie,S.Padiyar,R.Brundiers,T.Veit,J.Reintein, R.S.Goody,M.Konrad,I.Schlichting

Key ref:

N.Ostermann et al. (2000). Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP. J Mol Biol, 304, 43-53. PubMed id: 11071809 DOI: 10.1006/jmbi.2000.4175

Date:

10-Oct-00 Release date: 11-Oct-01

Protein chain

P23919  (KTHY_HUMAN) -  Thymidylate kinase from Homo sapiens

Seq: 212 a.a.

Struc: 210 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

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

dTMP + ATP (Bound ligand (Het Group name = ATM) matches with 83.33% similarity) = dTDP (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP

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

reference

DOI no: 10.1006/jmbi.2000.4175

J Mol Biol 304:43-53 (2000)

PubMed id: 11071809

Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP.

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

ABSTRACT

The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.

Selected figure(s)

Figure 3.
Figure 3. The 60-fold reduced catalytic rate with AZTMP in comparison to TMP suggests that TMPK can nevertheless adopt the partially closed (i.e. active) conformation despite the presence of the azido group. Overlay of the bisubstrate inhibitor complexes with either TP[5]A, shown in pink and modeled as TMP and ATP, and AZTP[5]A (modeled as AZTMP and ATP) reveal that the side-chain of Asp15 points away from the 3' substituent in the case of AZTP[5]A, but makes an interaction with the 3'-hydroxyl group in the case of TP[5]A. Only the fully closed conformation was observed for the P-loop in the presence of TP[5]A, while both the open and closed P-loop conformations were observed in the presence of AZTP[5]A, again suggesting a higher barrier for reaching the closed conformation in the presence of the azido group.

Figure 4.
Figure 4. The F105Y mutant adopts a partially closed conformation even in the presence of ADP. (a) Stereoview of the overlay between the complex of TMP and ADP with wild-type TMPK and the F105Y mutant, respectively. In the wild-type complex structure (depicted in pink), seven interconnected water molecules are observed to stabilize the open conformation. The presence of the hydroxyl group of Y105 hinders the formation of such a water structure, thereby destabilizing the open conformation. In addition, the hydroxyl moiety interacts with the side-chain of Gln157, which interacts with the amide nitrogen atom of the P-loop Asp15 (broken lines), resulting in the stabilization of the closed conformation. (b) To illustrate the steric clash that would ensue due to the introduced tyrosine hydroxyl moiety (instead of Phe), the structure observed in the open TMP-ADP complex is depicted with its interconnecting water structure, and the tyrosine residue (pink) from the F105Y structure.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 304, 43-53) copyright 2000.

Figures were selected by an automated process.