TRYPANOSOMA-CRUZI HAS NOT LAST 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 assume d to depend on putrescine uptake and S-adenosylmethionine decarboxylas e (AdoMetDC) for their synthesis of the polyamines spermidine and sper mine. 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 amp lified DNA fragment was used as a probe to isolate the complete AdoMet DC 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 AdoMetD C, the major differences being additional amino acids present in the t erminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetD Cs. When the coding region was expressed in Escherichia coli, the reco mbinant protein underwent autocatalytic cleavage, generating a 33-34 k Da alpha subunit and a 9 kDa beta subunit. The encoded protein catalys ed the decarboxylation of AdoMet (K-m 0.21 mM) and was stimulated by p utrescine but inhibited by the polyamines, weakly by spermidine and st rongly by spermine. Methylglyoxal-bis-(guanylhydrazone) (MGBG), a pote nt inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi e nzyme. This differential sensitivity to MGBG suggests that the two enz ymes are sufficiently different to warrant the search for compounds th at might interfere with the progression of Chagas' disease by selectiv ely inhibiting T. cruzi AdoMetDC.