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Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major

DOI: 10.1371/journal.pntd.0003781 DOI Help
PMID: 25978379 PMID Help

Authors: Jennifer Timm (University of York) , Cristina Bosch-navarrete (Consejo Superior de Investigaciones Científicas) , Eliseo Recio (Consejo Superior de Investigaciones Científicas) , Joanne E. Nettleship (Rutherford Appleton Laboratory) , Heather Rada (Rutherford Appleton Laboratory) , Dolores González-pacanowska (Consejo Superior de Investigaciones Científicas) , Keith Wilson (University of York) , Reza Salavati
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos Neglected Tropical Diseases , VOL 9 (5)

State: Published (Approved)
Published: May 2015
Diamond Proposal Number(s): 7864

Open Access Open Access

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.

Subject Areas: Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography