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Human CTP synthetase 2 - glutaminase domain in complex with 5-OXO-L-NORLEUCINE

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PDB Code 2V4U Target Class Nucleotide metabolism Target CTPS2 Alias CTPS2, DKFZp686C17207, FLJ43358, MGC32997 Disease Area/Function cancer Date Deposited Sep 29 2008 Authors M.WELIN, M.MOCHE, J.ANDERSSON, C.H.ARROWSMITH, H.BERGLUND, 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, K.OLESEN, C.PERSSON, J.SAGEMARK, H.SCHUELER, A.G.THORSELL, L.TRESAUGUES, S.VAN DEN BERG, M.WISNIEWSKA, M.WIKSTROM, P.NORDLUND Related Structure 2VKT, 3IHL

About this structure

CTP synthetase (CTPS, EC 6.3.4.2) 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 (1). 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 (2).

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

Cancer cells that exhibit increased cell proliferation also exhibit an increased activity of CTP synthetase. Thus, CTP synthetase is an attractive target for selective chemotherapy (3). There are several glutamine analogs that are known to inactivate several amidotransferases, like acivicin, azaserine and 6-Diazo-5-oxo-L-norleucine (DON). There are two isoforms of the human CTPS, CTPS and CTPS2, where CTPS2 is slightly smaller than CTPS and has a sequence identity of 74% (4). The structures of the full length E. coli CTPS and the synthetase domain of human CTPS (5) build up homotetramers while the glutaminase domains of E. coli CTPS are monomers (2).

Apo crystals of the glutaminase domain of CTPS2 were soaked with DON and a data set to 2.3 Å resolution was collected. The structure of CTPS2 soaked with DON was solved using molecular replacement using the glutaminase domain of the human CTPS2 as a search model. The glutamine analog was found to be covalently bound to the catalytically active Cys399. This observation has also been observed for other amidotransferases like glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) [6]. The inactivated form of the glutaminase domain of CTPS2 is the first CTPS with a glutamine analog bound. The structural information gained here can be used to design more specific inhibitors of CTPS.

Follow-up structure of 2VKT.

References

  1. Kent C, Carman GM. Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis and secretion through the Golgi apparatus.Trends Biochem Sci. 1999 Apr;24(4):146-50.
  2. Endrizzi JA, Kim H, Anderson PM, Baldwin EP. 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. 2004 Jun 1;43(21):6447-63.
  3. Kizaki H, Williams JC, Morris HP, Weber G. Increased cytidine 5'-triphosphate synthetase activity in rat and human tumors. Cancer Res. 1980 Nov;40(11):3921-7.
  4. van Kuilenburg AB, Meinsma R, Vreken P, Waterham HR, van Gennip AH. Identification of a cDNA encoding an isoform of human CTP synthetase. Biochim Biophys Acta. 2000 Jul 24;1492(2-3):548-52.
  5. Kursula P, Flodin S, Ehn M, Hammarström M, Schüler H, Nordlund P, Stenmark P. Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Jul 1;62(Pt 7):613-7.
  6. Krahn JM, Kim JH, Burns MR, Parry RJ, Zalkin H, Smith JL. Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site. Biochemistry. 1997 Sep 16;36(37):11061-8.