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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.


Non-reducing SDS-PAGE gels showing the effect of DTT and hydroperoxide treatments on MfOhrdel (A), MfOhrdel C154S (B) and MfOhrdel C87S (C). 10 µM of each protein were incubated during 1 h at 37 °C with 10 mM DTT, 0.1 mM H2O2, CuOOH or tBOOH or 17 µM linoleic acid hydroperoxide (LAOOH). All reactions were carried out in a buffer containing 0.5 M NaCl, 20 mM sodium phosphate pH 7.4 and 1 mM DTPA. Immediately after DTT or hydroperoxides treatments, all the samples were alkylated with NEM (100 mM) for 1 h at room temperature to avoid oxidation artefacts due to protein denaturation by SDS.
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f0050: Non-reducing SDS-PAGE gels showing the effect of DTT and hydroperoxide treatments on MfOhrdel (A), MfOhrdel C154S (B) and MfOhrdel C87S (C). 10 µM of each protein were incubated during 1 h at 37 °C with 10 mM DTT, 0.1 mM H2O2, CuOOH or tBOOH or 17 µM linoleic acid hydroperoxide (LAOOH). All reactions were carried out in a buffer containing 0.5 M NaCl, 20 mM sodium phosphate pH 7.4 and 1 mM DTPA. Immediately after DTT or hydroperoxides treatments, all the samples were alkylated with NEM (100 mM) for 1 h at room temperature to avoid oxidation artefacts due to protein denaturation by SDS.

Mentions: We next studied the thiol redox state of Cys residue in response to hydroperoxides by non-reducing SDS-PAGE, since the intramolecular disulfide bond of Ohr enzymes can be detected due to its lower hydrodynamic volume as a band (band b) that migrates faster than the reduced state (band a) [14]. Wt, C87S and C154S MfOhr were exposed to reducing (10 mM of DTT) or oxidative conditions (0.1 mM of CuOOH, tBOOH or H2O2 and 0.017 mM of LAOOH) during 1 h at 37 °C. For the Wt MfOhr, we observed the appearance of band b upon oxidation as expected since it corresponds to the intramolecular disulfide (Fig. 10A). Band b was not observed when Cp or Cr residues were independently substituted by serine residues. In this case, a single band (band a) was observed that migrated equally regardless of conditions (Fig. 10B and C).


Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi
Non-reducing SDS-PAGE gels showing the effect of DTT and hydroperoxide treatments on MfOhrdel (A), MfOhrdel C154S (B) and MfOhrdel C87S (C). 10 µM of each protein were incubated during 1 h at 37 °C with 10 mM DTT, 0.1 mM H2O2, CuOOH or tBOOH or 17 µM linoleic acid hydroperoxide (LAOOH). All reactions were carried out in a buffer containing 0.5 M NaCl, 20 mM sodium phosphate pH 7.4 and 1 mM DTPA. Immediately after DTT or hydroperoxides treatments, all the samples were alkylated with NEM (100 mM) for 1 h at room temperature to avoid oxidation artefacts due to protein denaturation by SDS.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0050: Non-reducing SDS-PAGE gels showing the effect of DTT and hydroperoxide treatments on MfOhrdel (A), MfOhrdel C154S (B) and MfOhrdel C87S (C). 10 µM of each protein were incubated during 1 h at 37 °C with 10 mM DTT, 0.1 mM H2O2, CuOOH or tBOOH or 17 µM linoleic acid hydroperoxide (LAOOH). All reactions were carried out in a buffer containing 0.5 M NaCl, 20 mM sodium phosphate pH 7.4 and 1 mM DTPA. Immediately after DTT or hydroperoxides treatments, all the samples were alkylated with NEM (100 mM) for 1 h at room temperature to avoid oxidation artefacts due to protein denaturation by SDS.
Mentions: We next studied the thiol redox state of Cys residue in response to hydroperoxides by non-reducing SDS-PAGE, since the intramolecular disulfide bond of Ohr enzymes can be detected due to its lower hydrodynamic volume as a band (band b) that migrates faster than the reduced state (band a) [14]. Wt, C87S and C154S MfOhr were exposed to reducing (10 mM of DTT) or oxidative conditions (0.1 mM of CuOOH, tBOOH or H2O2 and 0.017 mM of LAOOH) during 1 h at 37 °C. For the Wt MfOhr, we observed the appearance of band b upon oxidation as expected since it corresponds to the intramolecular disulfide (Fig. 10A). Band b was not observed when Cp or Cr residues were independently substituted by serine residues. In this case, a single band (band a) was observed that migrated equally regardless of conditions (Fig. 10B and C).

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.