Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme.
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.
Persson
K
K
Department of Cellular and Developmental Biology, Umeâ University, S-901 87 Umeå, Sweden.
Aslund
L
L
Grahn
B
B
Hanke
J
J
Heby
O
O
D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D007700:Kinetics; D008935:Mitoguazone / Q000494:pharmacology; D008969:Molecular Sequence Data; D011700:Putrescine / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013096:Spermine; D012437:Adenosylmethionine Decarboxylase; D008935:Mitoguazone; D013095:Spermidine; D011700:Putrescine