The glucose-6-phosphate dehydrogenase from Trypanosoma cruzi: its role in the defense of the parasite against oxidative stress.
The Trypanosoma cruzi glucose-6-phosphate dehydrogenase (G6PDH) is encoded by several genes located in three of the parasite chromosomes. All the sequences present two possible start codons, 111bp apart, also present in its Trypanosoma brucei counterpart. As the 37 residues comprised between the two candidate initiator methionines of T. brucei and T. cruzi G6PDHs constitute an unusual N-terminal extension only present in trypanosomatids, two forms of the T. cruzi G6PDH were expressed in Escherichia coli: a long one (Tc-G6PDH-L) translated from the first ATG codon, and a short one (Tc-G6PDH-S) translated from the second. Both were purified and their kinetic constants determined. The apparent K(m) for glucose-6-phosphate was 189.9, 98.4, and 288microM, for Tc-G6PDH-L, Tc-G6PDH-S and native Tc-G6PDH, respectively. The apparent K(m) for NADP was similar for both recombinant proteins. The Tc-G6PDH-L as well as the native enzyme, was inactivated by DTT while the Tc-G6PDH-S was unaffected by the reducing agent. This behavior could be related to the presence of two Cys groups in the N-terminal extension of the Tc-G6PDH-L similarly to the redox regulated G6PDHs from chloroplasts and cyanobacteria. This property, together with a remarkable induction (up to 46-fold) of the T. cruzi G6PDH in metacyclic trypomastigotes under oxidative stress conditions, suggests that the enzyme may play a prominent role in the defense mechanisms of the parasite against oxidative stress becoming an important target for chemotherapy. Western blots using antibodies against the N-terminal extension in Tc-G6PDH-L show that this form is expressed in the parasite.
Igoillo-Esteve
Mariana
M
Instituto de Investigaciones Biotecnológicas/INTECH, Universidad Nacional de General San Martin/CONICET, Av. General Paz s/n, INTI, Edificio 24, 1650 San Martin, Buenos Aires, Argentina.
Cazzulo
Juan José
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017125:Genes, Protozoan; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D016054:DNA, Protozoan; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase