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Trypanosoma brucei CTP synthetase - glutaminase domain in complex with acivicin

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PDB Code 2W7T Target Class Nucleotide metabolism Target TBCTPS Alias n/a Disease Area/Function parasitic disease Date Deposited Dec 30 2008 Authors M.Welin, M.Moche, C.H.Arrowsmith, H.Berglund, C.Bountra, R.Collins, L.G.Dahlgren, A.M.Edwards, S.Flodin, A.Flores, S.Graslund, M.Hammarstrom, A.Johansson, I.Johansson, T.Karlberg, T.Kotenyova, L.Lehtio, M.E.Nilsson, T.Nyman, C.Persson, J.Sagemark, H.Schueler, M.I.Siponen, A.G.Thorsell, L.Tresaugues, S.Van Den Berg, J.Weigelt, M.Wikstrom, M.Wisniewska, P.Nordlund.

About this structure

The parasite Trypanosoma brucei is causing the lethal disease, African sleeping sickness. Low CTP pools in comparison with mammalian cells and the inability to salvage cytosine and cytidine makes the enzyme CTP synthetase (CTPS, EC 6.3.4.2) a potential drug target in trypanosomes [1,2]. CTP synthetase is a rate-limiting enzyme in the synthesis of cytosine nucleotides, which play an important role in various metabolic processes and provide the precursors necessary for the synthesis of RNA and DNA [3]. CTPS is built up of two domains, a synthetase domain and a glutaminase domain. CTPS catalyzes the formation of CTP from UTP with the concomitant dephosphorylation of ATP and the deamination of glutamine to glutamate. The generated ammonia is transferred through a molecular tunnel to the synthetase domain [4]. CTPS belongs to the class 1 glutamine dependent amidotranferases and has a conserved catalytic triad consisting of a Cys-His-Glu [5]. The corresponding residues of the catalytic triad in TBCTPS are Cys419, His549 and Glu551.

ATP + UTP + NH3 => ADP + phosphate + CTP

There are several glutamine analogs like acivicin, azaserine and 6-Diazo-5-oxo-L-norleucine (DON) that are known to inactivate several amidotransferases. CTPS is a potential drug target in parasites and it has been shown that glutamine analogs suppress trypanosome proliferation in Trypanosoma brucei-infected mice [6].

Here we have determined the structure of the glutaminase domain of CTPS from Trypanosoma brucei (TBCTPS). The protein was pre-incubated with the glutamine analog acivicin prior to crystallization, and the analog was visible in the electron density covalently bound to Cys419. The glutaminase domain of TBCTPS is built up by a seven stranded β-sheet surrounded primarily by α-helices. This is the first structure of a CTPS with acivicin bound and the structural information gained will aid future drug design.

References

  1. Hofer A, Steverding D, Chabes A, Brun R, Thelander L. (2001) Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness. Proc Natl Acad Sci U S A. 22;98(11):6412-6.
  2. Hofer A, Ekanem JT, Thelander L. (1998) Allosteric regulation of Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo. J. Biol. Chem 273, 34098-34104.
  3. Kent C, Carman GM. (1999) Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis and secretion through the Golgi apparatus.Trends Biochem Sci. Apr;24(4):146-50.
  4. Endrizzi JA, Kim H, Anderson PM, Baldwin EP. (2004) Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets. Biochemistry. Jun 1;43(21):6447-63.
  5. Tesmer JJ, Klem TJ, Deras ML, Davisson VJ, Smith JL. (1996) The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families. Nat Struct Biol. Jan;3(1):74-86.
  6. Fijolek A, Hofer A, Thelander L. (2007) Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness. J Biol Chem. Apr 20;282(16):11858-65.