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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.


Comparison of the peroxidase activities of MfOhrdel and the C87S and C154S mutants. A. Lipoamide/lipoamide dehydrogenase coupled assay. The reactions were performed at 37 °C with 1 µM MfOhrdel (blue line) or 10 µM mutant proteins (C87S, red line or C154S, green line), in the presence of 50 µM reduced lipoamide, 100 µM DTPA, 0.5 µM XfLpd and 200 µM NADH in 50 µM sodium phosphate pH 7.4. Reactions were initiated by addition of 200 µM CuOOH. Blank reaction (black line) was performed without enzyme. B. The consumption of CuOOH was monitored during 8 min using FOX assay. The reactions were carried out in the presence of 1 µM (MfOhrdel) or 10 µM (C154S or C86S) enzymes. The control reactions for each tested hydroperoxide (enzyme+peroxide without DTT) and (hydroperoxide+DTT without enzyme) are not showed here. The figure is representative of at least two independent sets of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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f0040: Comparison of the peroxidase activities of MfOhrdel and the C87S and C154S mutants. A. Lipoamide/lipoamide dehydrogenase coupled assay. The reactions were performed at 37 °C with 1 µM MfOhrdel (blue line) or 10 µM mutant proteins (C87S, red line or C154S, green line), in the presence of 50 µM reduced lipoamide, 100 µM DTPA, 0.5 µM XfLpd and 200 µM NADH in 50 µM sodium phosphate pH 7.4. Reactions were initiated by addition of 200 µM CuOOH. Blank reaction (black line) was performed without enzyme. B. The consumption of CuOOH was monitored during 8 min using FOX assay. The reactions were carried out in the presence of 1 µM (MfOhrdel) or 10 µM (C154S or C86S) enzymes. The control reactions for each tested hydroperoxide (enzyme+peroxide without DTT) and (hydroperoxide+DTT without enzyme) are not showed here. The figure is representative of at least two independent sets of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Mentions: To evaluate the catalytic role of Cys residues of MfOhr, we generated single mutants for each residue. Both mutants lost their peroxidase activity as assessed by lipoamide/lipoamide dehydrogenase coupled assay and FOX assay (Fig. 8). Similar results were observed for Ohr from X. fastidiosa[3]. These results suggest the Cr might have a role in activating Cp for hydroperoxide reduction.


Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi
Comparison of the peroxidase activities of MfOhrdel and the C87S and C154S mutants. A. Lipoamide/lipoamide dehydrogenase coupled assay. The reactions were performed at 37 °C with 1 µM MfOhrdel (blue line) or 10 µM mutant proteins (C87S, red line or C154S, green line), in the presence of 50 µM reduced lipoamide, 100 µM DTPA, 0.5 µM XfLpd and 200 µM NADH in 50 µM sodium phosphate pH 7.4. Reactions were initiated by addition of 200 µM CuOOH. Blank reaction (black line) was performed without enzyme. B. The consumption of CuOOH was monitored during 8 min using FOX assay. The reactions were carried out in the presence of 1 µM (MfOhrdel) or 10 µM (C154S or C86S) enzymes. The control reactions for each tested hydroperoxide (enzyme+peroxide without DTT) and (hydroperoxide+DTT without enzyme) are not showed here. The figure is representative of at least two independent sets of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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f0040: Comparison of the peroxidase activities of MfOhrdel and the C87S and C154S mutants. A. Lipoamide/lipoamide dehydrogenase coupled assay. The reactions were performed at 37 °C with 1 µM MfOhrdel (blue line) or 10 µM mutant proteins (C87S, red line or C154S, green line), in the presence of 50 µM reduced lipoamide, 100 µM DTPA, 0.5 µM XfLpd and 200 µM NADH in 50 µM sodium phosphate pH 7.4. Reactions were initiated by addition of 200 µM CuOOH. Blank reaction (black line) was performed without enzyme. B. The consumption of CuOOH was monitored during 8 min using FOX assay. The reactions were carried out in the presence of 1 µM (MfOhrdel) or 10 µM (C154S or C86S) enzymes. The control reactions for each tested hydroperoxide (enzyme+peroxide without DTT) and (hydroperoxide+DTT without enzyme) are not showed here. The figure is representative of at least two independent sets of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Mentions: To evaluate the catalytic role of Cys residues of MfOhr, we generated single mutants for each residue. Both mutants lost their peroxidase activity as assessed by lipoamide/lipoamide dehydrogenase coupled assay and FOX assay (Fig. 8). Similar results were observed for Ohr from X. fastidiosa[3]. These results suggest the Cr might have a role in activating Cp for hydroperoxide reduction.

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.