title: Role of Δ1-pyrroline-5-carboxylate dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi.
abstract: 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 as follows: the conversion of proline to Δ(1)-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ(1)-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, two-dimensional gel electrophoresis, and small angle x-ray scattering 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, and 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 up-regulation (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.
authors.affiliation: From the Instituto de Ciências Biomédicas, Departamento de Parasitologia, Universidade de São Paulo, 05508-000 São Paulo, Brazil.
authors.forename: Brian S
authors.initials: BS
authors.lastname: Mantilla
authors.affiliation: From the Instituto de Ciências Biomédicas, Departamento de Parasitologia, Universidade de São Paulo, 05508-000 São Paulo, Brazil.
authors.forename: Lisvane S
authors.initials: LS
authors.lastname: Paes
authors.affiliation: From the Instituto de Ciências Biomédicas, Departamento de Parasitologia, Universidade de São Paulo, 05508-000 São Paulo, Brazil.
authors.forename: Elizabeth M F
authors.initials: EM
authors.lastname: Pral
authors.affiliation: the Laboratório de Biologia Estrutural, Instituto de Física de São Carlos, and.
authors.forename: Daiana E
authors.initials: DE
authors.lastname: Martil
authors.affiliation: the Laboratório de Biologia Estrutural, Instituto de Física de São Carlos, and.
authors.forename: Otavio H
authors.initials: OH
authors.lastname: Thiemann
authors.affiliation: the Departamento de Bioquímica, Biología Molecular y Celular, Universidad de Zaragoza, 50013 Zaragoza Spain.
authors.forename: Patricio
authors.initials: P
authors.lastname: Fernández-Silva
authors.affiliation: Instituto de Química, Departamento de Química Fundamental, Universidade de São Paulo, 13560-590 São Paulo, Brazil, and.
authors.forename: Erick L
authors.initials: EL
authors.lastname: Bastos
authors.affiliation: From the Instituto de Ciências Biomédicas, Departamento de Parasitologia, Universidade de São Paulo, 05508-000 São Paulo, Brazil, asilber@usp.br.
authors.forename: Ariel M
authors.initials: AM
authors.lastname: Silber
mesh_terms: D050842:1-Pyrroline-5-Carboxylate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014345:Trypanosoma / Q000472:pathogenicity*; D015854:Up-Regulation
chemical_list: D017931:DNA Primers; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase
keywords: Bioenergetics; Enzyme Mechanism; Host-Pathogen Interaction; Mitochondrial Metabolism; Parasite Metabolism; Trypanosoma cruzi