Role of {Delta}1-Pyrroline-5-Carboxylate-dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi [Bioenergetics]

January 26th, 2015 by Mantilla, B. S., Paes, L. S., Pral, E. M. F., Martil, D. E., Thiemann, O. H., Fernandez–Silva, P., Bastos, E. L., Silber, A. M.

Proline is crucial for energizing critical events throughout the life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease. The proline breakdown pathway consists of two oxidation steps, both of which produce reducing equivalents: the conversion of proline to Δ1-pyrroline-5-carboxylate (P5C) and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ1-Pyrroline-5-Carboxylate dehydrogenase from T. cruzi (TcP5CDH) and report here on how this enzyme contributes to a central metabolic pathway in this parasite. Size-exclusion chromatography, 2D-gel electrophoresis and SAXS analysis of TcP5CDH revealed an oligomeric state composed of two subunits of six protomers. TcP5CDH was found to complement a yeast strain deficient in PUT2 activity, confirming the enzyme's functional role, and the biochemical parameters (Km, kcat, kcat/Km) of the recombinant-TcP5CDH were determined, exhibiting values comparable with those from T. cruzi lysates. In addition, TcP5CDH exhibited mitochondrial staining during the main stages of the T. cruzi life cycle. mRNA and enzymatic activity levels indicated the upregulation (6-fold change) of TcP5CDH during the infective stages of the parasite. The participation of P5C as an energy source was also demonstrated. Overall, we propose that this enzymatic step is crucial for the viability of both replicative and infective forms of T. cruzi.
  • Posted in Journal of Biological Chemistry, Publications
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