Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Aug 1;333(Pt 3):527–537. doi: 10.1042/bj3330527

Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme.

K Persson 1, L Aslund 1, B Grahn 1, J Hanke 1, O Heby 1
PMCID: PMC1219613  PMID: 9677309

Abstract

All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have now identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids. The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T. cruzi genomic library. The AdoMetDC gene was located on chromosomes with a size of approx. 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues. The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs. When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit. The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine. Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi enzyme. This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T. cruzi AdoMetDC.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ariyanayagam M. R., Fairlamb A. H. Diamine auxotrophy may be a universal feature of Trypanosoma cruzi epimastigotes. Mol Biochem Parasitol. 1997 Jan;84(1):111–121. doi: 10.1016/s0166-6851(96)02788-0. [DOI] [PubMed] [Google Scholar]
  2. Bacchi C. J., Nathan H. C., Hutner S. H., McCann P. P., Sjoerdsma A. Polyamine metabolism: a potential therapeutic target in trypanosomes. Science. 1980 Oct 17;210(4467):332–334. doi: 10.1126/science.6775372. [DOI] [PubMed] [Google Scholar]
  3. Bacchi C. J., Yarlett N. Effects of antagonists of polyamine metabolism on African trypanosomes. Acta Trop. 1993 Sep;54(3-4):225–236. doi: 10.1016/0001-706x(93)90095-s. [DOI] [PubMed] [Google Scholar]
  4. Bangs J. D., Crain P. F., Hashizume T., McCloskey J. A., Boothroyd J. C. Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J Biol Chem. 1992 May 15;267(14):9805–9815. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Byers T. L., Bush T. L., McCann P. P., Bitonti A. J. Antitrypanosomal effects of polyamine biosynthesis inhibitors correlate with increases in Trypanosoma brucei brucei S-adenosyl-L-methionine. Biochem J. 1991 Mar 1;274(Pt 2):527–533. doi: 10.1042/bj2740527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cano M. I., Gruber A., Vazquez M., Cortés A., Levin M. J., González A., Degrave W., Rondinelli E., Zingales B., Ramirez J. L. Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi genome project. Mol Biochem Parasitol. 1995 May;71(2):273–278. doi: 10.1016/0166-6851(95)00066-a. [DOI] [PubMed] [Google Scholar]
  8. Cazzulo J. J., Franke de Cazzulo B. M., Engel J. C., Cannata J. J. End products and enzyme levels of aerobic glucose fermentation in trypanosomatids. Mol Biochem Parasitol. 1985 Sep;16(3):329–343. doi: 10.1016/0166-6851(85)90074-x. [DOI] [PubMed] [Google Scholar]
  9. De Lange T., Berkvens T. M., Veerman H. J., Frasch A. C., Barry J. D., Borst P. Comparison of the genes coding for the common 5' terminal sequence of messenger RNAs in three trypanosome species. Nucleic Acids Res. 1984 Jun 11;12(11):4431–4443. doi: 10.1093/nar/12.11.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fairlamb A. H., Blackburn P., Ulrich P., Chait B. T., Cerami A. Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science. 1985 Mar 22;227(4693):1485–1487. doi: 10.1126/science.3883489. [DOI] [PubMed] [Google Scholar]
  11. Fairlamb A. H., Cerami A. Metabolism and functions of trypanothione in the Kinetoplastida. Annu Rev Microbiol. 1992;46:695–729. doi: 10.1146/annurev.mi.46.100192.003403. [DOI] [PubMed] [Google Scholar]
  12. Frostesjö L., Holm I., Grahn B., Page A. W., Bestor T. H., Heby O. Interference with DNA methyltransferase activity and genome methylation during F9 teratocarcinoma stem cell differentiation induced by polyamine depletion. J Biol Chem. 1997 Feb 14;272(7):4359–4366. doi: 10.1074/jbc.272.7.4359. [DOI] [PubMed] [Google Scholar]
  13. Ghoda L., Phillips M. A., Bass K. E., Wang C. C., Coffino P. Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation. J Biol Chem. 1990 Jul 15;265(20):11823–11826. [PubMed] [Google Scholar]
  14. Goldberg B., Yarlett N., Sufrin J., Lloyd D., Bacchi C. J. A unique transporter of S-adenosylmethionine in African trypanosomes. FASEB J. 1997 Mar;11(4):256–260. doi: 10.1096/fasebj.11.4.9068614. [DOI] [PubMed] [Google Scholar]
  15. Hanke J., Sánchez D. O., Henriksson J., Aslund L., Pettersson U., Frasch A. C., Hoheisel J. D. Mapping the Trypanosoma cruzi genome: analyses of representative cosmid libraries. Biotechniques. 1996 Oct;21(4):686-8, 690-3. doi: 10.2144/96214rr01. [DOI] [PubMed] [Google Scholar]
  16. Hanson S., Adelman J., Ullman B. Amplification and molecular cloning of the ornithine decarboxylase gene of Leishmania donovani. J Biol Chem. 1992 Feb 5;267(4):2350–2359. [PubMed] [Google Scholar]
  17. Heby O., Persson L. Molecular genetics of polyamine synthesis in eukaryotic cells. Trends Biochem Sci. 1990 Apr;15(4):153–158. doi: 10.1016/0968-0004(90)90216-x. [DOI] [PubMed] [Google Scholar]
  18. Henriksson J., Aslund L., Macina R. A., Franke de Cazzulo B. M., Cazzulo J. J., Frasch A. C., Pettersson U. Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi. Mol Biochem Parasitol. 1990 Sep-Oct;42(2):213–223. doi: 10.1016/0166-6851(90)90164-h. [DOI] [PubMed] [Google Scholar]
  19. Henriksson J., Aslund L., Pettersson U. Karyotype variability in Trypanosoma cruzi. Parasitol Today. 1996 Mar;12(3):108–114. doi: 10.1016/0169-4758(96)80670-3. [DOI] [PubMed] [Google Scholar]
  20. Henriksson J., Porcel B., Rydåker M., Ruiz A., Sabaj V., Galanti N., Cazzulo J. J., Frasch A. C., Pettersson U. Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi. Mol Biochem Parasitol. 1995 Jul;73(1-2):63–74. doi: 10.1016/0166-6851(95)00096-j. [DOI] [PubMed] [Google Scholar]
  21. Hunter K. J., Le Quesne S. A., Fairlamb A. H. Identification and biosynthesis of N1,N9-bis(glutathionyl)aminopropylcadaverine (homotrypanothione) in Trypanosoma cruzi. Eur J Biochem. 1994 Dec 15;226(3):1019–1027. doi: 10.1111/j.1432-1033.1994.t01-1-01019.x. [DOI] [PubMed] [Google Scholar]
  22. Kashiwagi K., Taneja S. K., Liu T. Y., Tabor C. W., Tabor H. Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase. J Biol Chem. 1990 Dec 25;265(36):22321–22328. [PubMed] [Google Scholar]
  23. Kierszenbaum F., Wirth J. J., McCann P. P., Sjoerdsma A. Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4278–4282. doi: 10.1073/pnas.84.12.4278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Le Quesne S. A., Fairlamb A. H. Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes. Biochem J. 1996 Jun 1;316(Pt 2):481–486. doi: 10.1042/bj3160481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Majumder S., Wirth J. J., Bitonti A. J., McCann P. P., Kierszenbaum F. Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi. J Parasitol. 1992 Apr;78(2):371–374. [PubMed] [Google Scholar]
  26. Marić S. C., Crozat A., Jänne O. A. Structure and organization of the human S-adenosylmethionine decarboxylase gene. J Biol Chem. 1992 Sep 15;267(26):18915–18923. [PubMed] [Google Scholar]
  27. Murray V. Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 1989 Nov 11;17(21):8889–8889. doi: 10.1093/nar/17.21.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murray V. Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 1989 Nov 11;17(21):8889–8889. doi: 10.1093/nar/17.21.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pegg A. E., McCann P. P. S-adenosylmethionine decarboxylase as an enzyme target for therapy. Pharmacol Ther. 1992 Dec;56(3):359–377. doi: 10.1016/0163-7258(92)90025-u. [DOI] [PubMed] [Google Scholar]
  30. Perry K. L., Watkins K. P., Agabian N. Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8190–8194. doi: 10.1073/pnas.84.23.8190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ruan H., Shantz L. M., Pegg A. E., Morris D. R. The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element. J Biol Chem. 1996 Nov 22;271(47):29576–29582. doi: 10.1074/jbc.271.47.29576. [DOI] [PubMed] [Google Scholar]
  32. Sakai T., Hori C., Kano K., Oka T. Purification and characterization of S-adenosyl-L-methionine decarboxylase from mouse mammary gland and liver. Biochemistry. 1979 Dec 11;18(25):5541–5548. doi: 10.1021/bi00592a003. [DOI] [PubMed] [Google Scholar]
  33. Stanley B. A., Pegg A. E. Amino acid residues necessary for putrescine stimulation of human S-adenosylmethionine decarboxylase proenzyme processing and catalytic activity. J Biol Chem. 1991 Oct 5;266(28):18502–18506. [PubMed] [Google Scholar]
  34. Stanley B. A., Pegg A. E., Holm I. Site of pyruvate formation and processing of mammalian S-adenosylmethionine decarboxylase proenzyme. J Biol Chem. 1989 Dec 15;264(35):21073–21079. [PubMed] [Google Scholar]
  35. Stanley B. A., Shantz L. M., Pegg A. E. Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli. Determination of sites for putrescine activation of activity and processing. J Biol Chem. 1994 Mar 18;269(11):7901–7907. [PubMed] [Google Scholar]
  36. Tabor C. W., Tabor H. The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase. J Biol Chem. 1987 Nov 25;262(33):16037–16040. [PubMed] [Google Scholar]
  37. Taelman H., Schechter P. J., Marcelis L., Sonnet J., Kazyumba G., Dasnoy J., Haegele K. D., Sjoerdsma A., Wery M. Difluoromethylornithine, an effective new treatment of Gambian trypanosomiasis. Results in five patients. Am J Med. 1987 Mar 23;82(3 Spec No):607–614. doi: 10.1016/0002-9343(87)90107-0. [DOI] [PubMed] [Google Scholar]
  38. Tekwani B. L., Bacchi C. J., Pegg A. E. Putrescine activated S-adenosylmethionine decarboxylase from Trypanosoma brucei brucei. Mol Cell Biochem. 1992 Nov 4;117(1):53–61. doi: 10.1007/BF00230410. [DOI] [PubMed] [Google Scholar]
  39. Van Nieuwenhove S., Schechter P. J., Declercq J., Boné G., Burke J., Sjoerdsma A. Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL-alpha-difluoromethylornithine), an inhibitor of ornithine decarboxylase; first field trial. Trans R Soc Trop Med Hyg. 1985;79(5):692–698. doi: 10.1016/0035-9203(85)90195-6. [DOI] [PubMed] [Google Scholar]
  40. Williams-Ashman H. G., Schenone A. Methyl glyoxal bis(guanylhydrazone) as a potent inhibitor of mammalian and yeast S-adenosylmethionine decarboxylases. Biochem Biophys Res Commun. 1972 Jan 14;46(1):288–295. doi: 10.1016/0006-291x(72)90661-4. [DOI] [PubMed] [Google Scholar]
  41. Xiong H., Stanley B. A., Tekwani B. L., Pegg A. E. Processing of mammalian and plant S-adenosylmethionine decarboxylase proenzymes. J Biol Chem. 1997 Nov 7;272(45):28342–28348. doi: 10.1074/jbc.272.45.28342. [DOI] [PubMed] [Google Scholar]
  42. Yakubu M. A., Basso B., Kierszenbaum F. DL-alpha-difluoromethylarginine inhibits intracellular Trypanosoma cruzi multiplication by affecting cell division but not trypomastigote-amastigote transformation. J Parasitol. 1992 Jun;78(3):414–419. [PubMed] [Google Scholar]
  43. Yakubu M. A., Majumder S., Kierszenbaum F. Inhibition of S-adenosyl-L-methionine (AdoMet) decarboxylase by the decarboxylated AdoMet analog 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811) decreases the capacities of Trypanosoma cruzi to infect and multiply within a mammalian host cell. J Parasitol. 1993 Aug;79(4):525–532. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES