Authors

L Marchese¹; B S Mantilla¹; K Olavarría¹; A M Silber¹;  ¹ Biomedical Science Institute - USP, Brazil

Discussion

Mitochondrion of insect trypanosomes are able to oxidize L-proline (Pro) to L-glutamate (Glu) through two enzymatic steps for energy production. However, little is known about Pro biosynthesis in these organisms. Pro could be only produced from Glu by two reductions steps via Δ¹-pyrroline-5-carboxylate (P5C) synthase (P5CS) and P5C reductase (P5CR). Here, we show that the Glu-P5C-Pro pathway is operative in T. cruzi, and is absent in T. brucei. After depletion of the intracellular pools of free Pro, epimastigotes of T. cruzi were able to restore their Pro levels when supplied with Glu or P5C, while procyclics of T. brucei (PCFs) were not. Furthermore, neither P5CS nor P5CR were detected in PCF lysates using antibodies raised against both T. cruzi enzymes. Digitonin permeabilization and immunofluorescence of T. cruzi epimastigotes showed that P5CS and P5CR are cytosolic. A biochemical characterization of the recombinant TcP5CR was performed. It is a NADPH-dependent enzyme and this cofactor has a substrate-inhibitory effect on TcP5CR (K_i^app = 50,5 µM), suggesting that this enzyme is important in the regulation of Pro metabolism. Our results hint that Pro is not an essential metabolite in T. cruzi, while seems to be essential in T. brucei. Other possible roles for this metabolic pathway in T. cruzi are being explored.