title: Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi.
abstract: Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by β-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×108M-1s-1). Both Cys87and Cys154were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKavalue of the Cyspresidue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria.
authors.affiliation: Departmento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil. Electronic address: meireles@ib.usp.br.
authors.forename: D A
authors.initials: DA
authors.lastname: Meireles
authors.affiliation: Departmento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: R M
authors.initials: RM
authors.lastname: Domingos
authors.affiliation: Departmento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: J W
authors.initials: JW
authors.lastname: Gaiarsa
authors.affiliation: Departmento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: E G
authors.initials: EG
authors.lastname: Ragnoni
authors.affiliation: Departmento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: R
authors.initials: R
authors.lastname: Bannitz-Fernandes
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: J F
authors.initials: JF
authors.lastname: da Silva Neto
authors.affiliation: Departmento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: R F
authors.initials: RF
authors.lastname: de Souza
authors.affiliation: Departmento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil. Electronic address: nettoles@ib.usp.br.
authors.forename: L E S
authors.initials: LES
authors.lastname: Netto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D056890:Eukaryota / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D028521:Musa / Q000382:microbiology; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
chemical_list: D005656:Fungal Proteins; D010544:Peroxidases; D003545:Cysteine
keywords: Ohr/OsmC; Phylogeny; Thiol-dependent peroxidases