title: Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding.
abstract: 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 (k(cat)) 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 evolution.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Farahnaz
authors.initials: F
authors.lastname: Ranjbarian
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Munender
authors.initials: M
authors.lastname: Vodnala
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Sharvani Munender
authors.initials: SM
authors.lastname: Vodnala
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden; Department of Biochemistry, Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, Tehran University, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Rofougaran
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Thelander
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. Electronic address: anders.hofer@medchem.umu.se.
authors.forename: Anders
authors.initials: A
authors.lastname: Hofer
mesh_terms: D000595:Amino Acid Sequence; D019143:Evolution, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D009711:Nucleotides; D015800:Protozoan Proteins; D013937:Thymidine Kinase