The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression

NADP-linked glutamate dehydrogenase (NADP⁺-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH₁ and TcGluDH₂) were isolated and sequenced. The sequences obtained are most similar to that of the NADP⁺-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH₁ as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP⁺-GluDH in different parasite stages was obtained. TcGluDH₁ encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.