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Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

View Article: PubMed Central - PubMed

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)×108 M−1 s−1). Both Cys87 and Cys154 were 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 pKa value of the Cysp residue 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.

No MeSH data available.


Western blots of total (TF), cytosolic (CF) and enriched mitochondria (EMF) fractions of protoplasts cells of M. fijiensis Mf_1. Fractions of M. fijiensis protoplasts are described in Material and Methods. A. Loading control (ponceau staining) of cellular fractions. After western blot, membrane was probed with affinity purified MfOhr polyclonal antibody (B); PGK-1, a cytoplasmic marker (C) and COX-IV, a mitochondrial marker (D), respectively.
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f0055: Western blots of total (TF), cytosolic (CF) and enriched mitochondria (EMF) fractions of protoplasts cells of M. fijiensis Mf_1. Fractions of M. fijiensis protoplasts are described in Material and Methods. A. Loading control (ponceau staining) of cellular fractions. After western blot, membrane was probed with affinity purified MfOhr polyclonal antibody (B); PGK-1, a cytoplasmic marker (C) and COX-IV, a mitochondrial marker (D), respectively.

Mentions: To experimentally verify if MfOhr is in fact a mitochondrial protein, we performed the subcellular fractionation of M. fijiensis protoplast cells, followed by western blot analysis. Initially, the affinity of purified Ohr antibody raised against bacterial Ohr was shown to be able to detect recombinant MfOhrdel (Fig. S2). However, MfOhr could not be detected in whole extracts of M. fijiensis mycelia grown in PDB medium, even when high amounts of total protein (200–300 µg) were employed. In contrast, a strong signal was observed in the enriched mitochondrial fraction (Fig. 11B). This is likely due to a dilution effect as the mitochondria occupy only a small fraction of the whole cellular mass. Indeed, COX IV, a well-established and abundant mitochondrial protein, was detected in whole extract at significantly lower levels than in the enriched mitochondrial fraction (Fig. 11D). As another control, the cytoplasmic protein PGK-1 was present in the total and cytoplasmic fraction but not in the mitochondrial fraction (Fig. 11C). Taken together, these results confirm the in silico prediction that MfOhr is a mitochondrial protein.


Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi
Western blots of total (TF), cytosolic (CF) and enriched mitochondria (EMF) fractions of protoplasts cells of M. fijiensis Mf_1. Fractions of M. fijiensis protoplasts are described in Material and Methods. A. Loading control (ponceau staining) of cellular fractions. After western blot, membrane was probed with affinity purified MfOhr polyclonal antibody (B); PGK-1, a cytoplasmic marker (C) and COX-IV, a mitochondrial marker (D), respectively.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5384416&req=5

f0055: Western blots of total (TF), cytosolic (CF) and enriched mitochondria (EMF) fractions of protoplasts cells of M. fijiensis Mf_1. Fractions of M. fijiensis protoplasts are described in Material and Methods. A. Loading control (ponceau staining) of cellular fractions. After western blot, membrane was probed with affinity purified MfOhr polyclonal antibody (B); PGK-1, a cytoplasmic marker (C) and COX-IV, a mitochondrial marker (D), respectively.
Mentions: To experimentally verify if MfOhr is in fact a mitochondrial protein, we performed the subcellular fractionation of M. fijiensis protoplast cells, followed by western blot analysis. Initially, the affinity of purified Ohr antibody raised against bacterial Ohr was shown to be able to detect recombinant MfOhrdel (Fig. S2). However, MfOhr could not be detected in whole extracts of M. fijiensis mycelia grown in PDB medium, even when high amounts of total protein (200–300 µg) were employed. In contrast, a strong signal was observed in the enriched mitochondrial fraction (Fig. 11B). This is likely due to a dilution effect as the mitochondria occupy only a small fraction of the whole cellular mass. Indeed, COX IV, a well-established and abundant mitochondrial protein, was detected in whole extract at significantly lower levels than in the enriched mitochondrial fraction (Fig. 11D). As another control, the cytoplasmic protein PGK-1 was present in the total and cytoplasmic fraction but not in the mitochondrial fraction (Fig. 11C). Taken together, these results confirm the in silico prediction that MfOhr is a mitochondrial protein.

View Article: PubMed Central - PubMed

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)×108 M−1 s−1). Both Cys87 and Cys154 were 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 pKa value of the Cysp residue 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.

No MeSH data available.