Trypanosoma brucei Thymidine Kinase Is Tandem Protein Consisting of Two Homologous Parts, Which Together Enable Efficient Substrate Binding

Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2012; 287(21):17628-36. DOI: 10.1074/jbc.M112.340059
Source: PubMed


Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy.
We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized.
Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine >
deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary
structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains
showed that only domain 2 was active. It had a similar turnover number (kcat) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity.
Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner
that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues
are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly
identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote

Download full-text


Available from: Anders Hofer, Jul 02, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Thymidine kinase 1 (TK1) provides a crucial precursor, thymidine monophosphate (dTMP), for nucleic acid synthesis, and the activity of TK1 increases up to 200-fold during the S-phase of cell division in humans. An important part of the regulatory check-points is the ATP and enzyme concentration-dependent transition of TK1 from a dimer with low catalytic efficiency to a tetramer with high catalytic efficiency. This regulatory fine-tuning serves as an additional control to provide the balanced pool of nucleic acid precursors in the cell. We sub-cloned and over-expressed ten different TK1s, originating from widely different organisms, and characterized their kinetic and oligomerization properties. While bacteria, plants and Dictyostelium only exhibited dimeric TK1, we found that all animals had a tetrameric TK1. However, a clear ATP dependent switch between dimer and tetramer was found only in higher vertebrates, and was especially pronounced in mammalian and bird TK1s. We suggest that the dimer form is the original one and the tetramer originated in the animal lineage after the split of Dictyostelium and the lineages leading to invertebrates and vertebrates. The efficient switching mechanism was likely settled first in the warm-blooded animals when they separated from the rest of vertebrates. © 2013 The Authors Journal compilation © 2013 FEBS.
    FEBS Journal 01/2013; 280(6). DOI:10.1111/febs.12154 · 4.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    PLoS Neglected Tropical Diseases 05/2015; 9(5):e0003781. DOI:10.1371/journal.pntd.0003781 · 4.45 Impact Factor