title: Biochemical characterization of stage-specific isoforms of aspartate aminotransferases from Trypanosoma cruzi and Trypanosoma brucei.
abstract: Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Trypanosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T. cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T. brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T. brucei ASATs were very similar to those determined for T. cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T. cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T. cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T. cruzi, and suggest that the succinate excreted by amastigotes more likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism.
authors.affiliation: Instituto de Química y Fisicoquímica Biológica IQUIFIB-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina.
authors.forename: Daniela
authors.initials: D
authors.lastname: Marciano
authors.forename: Constanza
authors.initials: C
authors.lastname: Llorente
authors.forename: Dante A
authors.initials: DA
authors.lastname: Maugeri
authors.forename: Candelaria
authors.initials: C
authors.lastname: de la Fuente
authors.forename: Fred
authors.initials: F
authors.lastname: Opperdoes
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Cazzulo
authors.forename: Cristina
authors.initials: C
authors.lastname: Nowicki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000096:biosynthesis; D008830:Microbodies / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity; D019802:Succinic Acid / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D007656:Ketoglutaric Acids; D001224:Aspartic Acid; D019802:Succinic Acid; D008291:Malate Dehydrogenase; D001219:Aspartate Aminotransferases