Premium
Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei
Author(s) -
Valente Maria,
Timm Jennifer,
CastilloAcosta Víctor M.,
RuizPérez Luis M.,
Balzarini Tom,
Nettleship Joanne E.,
Bird Louise E.,
Rada Heather,
Wilson Keith S.,
GonzálezPacanowska Dolores
Publication year - 2016
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13467
Subject(s) - biology , trypanosoma brucei , biochemistry , thymidine , enzyme , active site , thymidine kinase , thymine , nucleotide salvage , nucleoside , dna , nucleotide , virus , herpes simplex virus , gene , virology
Summary 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 ( Tb TK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that Tb TK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of Tb TK is cell cycle regulated and that depletion of Tb TK 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 Tb TK 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 Tb TK 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.