a cdna encoding pavine n-methyltransferase s-adenosylmethionine-dependent was heterologously expressed in escherichia coli the enzyme was purified usin
All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. It has been assumed that parasites depend on the uptake of putrescine and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to amino acid sequences preserved in AdoMetDC proteins from other trypanosomatides. 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 additional amino acid differences being present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found compared to AdoMetDCs trypanosomatid. 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 catalyzed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by 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.