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

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Available from: Anders Hofer, Jul 02, 2015
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