title: Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration.
abstract: BACKGROUND
  Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported.
  
  
  METHODS
  We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches.
  
  
  RESULTS
  Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO(-) molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.
  
  
  CONCLUSIONS
  These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.
  
  
  CONCLUSIONS
  This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.
authors.affiliation: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín (EEZ), CSIC, Apartado 419, E-18080 Granada, Spain. Electronic address: javier.corpas@eez.csic.es.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Corpas
authors.forename: Marina
authors.initials: M
authors.lastname: Leterrier
authors.forename: Juan C
authors.initials: JC
authors.lastname: Begara-Morales
authors.forename: Raquel
authors.initials: R
authors.lastname: Valderrama
authors.forename: Mounira
authors.initials: M
authors.lastname: Chaki
authors.forename: Javier
authors.initials: J
authors.lastname: López-Jaramillo
authors.forename: Francisco
authors.initials: F
authors.lastname: Luque
authors.forename: José M
authors.initials: JM
authors.lastname: Palma
authors.forename: María N
authors.initials: MN
authors.lastname: Padilla
authors.forename: Beatriz
authors.initials: B
authors.lastname: Sánchez-Calvo
authors.forename: Capilla
authors.initials: C
authors.lastname: Mata-Pérez
authors.forename: Juan B
authors.initials: JB
authors.lastname: Barroso
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D005978:Glutathione / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D050537:Hydroxypyruvate Reductase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D020675:Peroxisomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*
chemical_list: D009566:Nitrates; D020543:Proteome; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D006861:Hydrogen Peroxide; D050537:Hydroxypyruvate Reductase; D005978:Glutathione
keywords: 3-morpholinosydnonimine; GSH; GSNO; HPR; Hydroxypyruvate reductase; NO; Nitration; Nitric oxide; Nitro-proteome; ONOO(−); PTM; Peroxisome; Peroxynitrite; RNS; S-nitrosoglutathione; SIN-1; hydroxypyruvate reductase; nitric oxide; peroxynitrite; post-translational modification; reactive nitrogen species; reduced glutathione