Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi

A single form of serine hydroxymethyltransferase (SHMT) was detected in epi mastigotes of Trypanosoma cruzi, in contrast to the three isoforms of the e nzyme characterized from another trypanosomatid, Crithidia fasciculata [Cap elluto D.G.S., Hellman U., Cazzulo J.J. & Cannata J.J.B. (1999) Mol Biochem Parasitol, 98, 187-201]: The T. cruzi SHMT was found to be highly unstable in crude extracts. In the presence of the cysteine proteinase inhibitors N -alpha-p-tosyl-L-lysine chloromethyl ketone and L-trans-3-carboxyoxiran-2-c arbonyl-L-leucylagmatine, however, the enzyme could be purified to homogene ity. Digitonin treatment of intact cells suggested that the enzyme is cytos olic. T. cruzi SHMT presents a monomeric structure shown by the apparent mo lecular masses of 69 kDa (native) and 55 kDa (subunit) determined by Sephad ex G-200 gel filtration and SDS/PAGE, respectively, This is in contrast to the tetrameric SHMTs described in C. fasciculata and other eukaryotes. The enzyme was pyridoxal phosphate-dependent after L-cysteine and hydroxylamine treatments and it was strongly inhibited lay the substrate analog folate, which was competitive towards tetrahydrofolate and noncompetitive towards L -serine. Partial sequencing of tryptic internal peptides of the enzyme indi cate considerable similarity with other SHMTs, particularly from those of p lant origin.