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Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei

DOI: 10.1111/mmi.13467 DOI Help

Authors: Maria Valente (Instituto de Parasitología y Biomedicina “López-Neyra”, Consejo Superior de Investigaciones Científicas) , Jennifer Timm (University of York) , Víctor M. Castillo-acosta (Instituto de Parasitología y Biomedicina “López-Neyra”, Consejo Superior de Investigaciones Científicas) , Luis M. Ruiz-pérez (Instituto de Parasitología y Biomedicina “López-Neyra”, Consejo Superior de Investigaciones Científicas) , Tom Balzarini (Instituto de Parasitología y Biomedicina “López-Neyra”, Consejo Superior de Investigaciones Científicas) , Joanne E. Nettleship (The Oxford Protein Production Facility, Research Complex at Harwell, Rutherford Appleton Laboratory) , Louise E Bird (The Oxford Protein Production Facility, Research Complex at Harwell, Rutherford Appleton Laboratory) , Heather Rada (The Oxford Protein Production Facility, Research Complex at Harwell, Rutherford Appleton Laboratory) , Keith S. Wilson (York Structural Biology Laboratory, Department of Chemistry, University of York) , Dolores González-pacanowska (Instituto de Parasitología y Biomedicina “López-Neyra”, Consejo Superior de Investigaciones Científicas)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Molecular Microbiology

State: Published (Approved)
Published: July 2016
Diamond Proposal Number(s): 7864

Abstract: Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design. This article is protected by copyright. All rights reserved.

Subject Areas: Biology and Bio-materials, Chemistry, Medicine


Instruments: I02-Macromolecular Crystallography , I04-Macromolecular Crystallography