4UXI

Leishmania major Thymidine Kinase in complex with thymidine


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.74 Å
  • R-Value Free: 0.310 
  • R-Value Work: 0.282 
  • R-Value Observed: 0.284 

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This is version 1.2 of the entry. See complete history


Literature

Structural and Kinetic Characterization of Thymidine Kinase from Leishmania Major.

Timm, J.Bosch-Navarrete, C.Recio, E.Nettleship, J.E.Rada, H.Gonzalez-Pacanowska, D.Wilson, K.S.

(2015) PLoS Negl Trop Dis 9: 3781

  • DOI: https://doi.org/10.1371/journal.pntd.0003781
  • Primary Citation of Related Structures:  
    4UXH, 4UXI, 4UXJ

  • PubMed Abstract: 

    Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s(-1) and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.


  • Organizational Affiliation

    Structural Biology Laboratory, Department of Chemistry, University of York, York, United Kingdom.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
THYMIDINE KINASE
A, B
184Leishmania majorMutation(s): 0 
EC: 2.7.1.21
UniProt
Find proteins for Q4QC75 (Leishmania major)
Explore Q4QC75 
Go to UniProtKB:  Q4QC75
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ4QC75
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.74 Å
  • R-Value Free: 0.310 
  • R-Value Work: 0.282 
  • R-Value Observed: 0.284 
  • Space Group: P 65 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 121.149α = 90
b = 121.149β = 90
c = 116.22γ = 120
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2015-05-27
    Type: Initial release
  • Version 1.1: 2016-03-02
    Changes: Data collection
  • Version 1.2: 2024-05-08
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description