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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.


Genomic arrangements of genes of the Ohr/OsmC family present in eukaryotes.
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f0015: Genomic arrangements of genes of the Ohr/OsmC family present in eukaryotes.

Mentions: Several eukaryotic genomes present more than one member of the Ohr/OsmC family, being arranged in highly variable configurations (Fig. 3). For instance, some fungi microorganisms present two or three ohr paralogues in their genomes (Fig. 3A and B), some located near to each other, in some cases the two gene are even neighbors (Fig. 3A), suggesting the occurrence of gene duplication events. Besides fungi, microorganisms that contain more than one gene of the Ohr/OsmC family are: M. fijiensis presenting one ohr and one ohr-like gene (Fig. 3C); Trichomonas vaginalis with four ohr-like genes (as also described by [22]) and D. discoideum AX4 containing one copy of osmC and one copy of an ohr-like gene (Fig. 3D). On the other hand, most of the other genomes encode only a single homolog of the ohr subfamily, as is the case of the moss P. patens (Fig. 3E).


Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi
Genomic arrangements of genes of the Ohr/OsmC family present in eukaryotes.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: Genomic arrangements of genes of the Ohr/OsmC family present in eukaryotes.
Mentions: Several eukaryotic genomes present more than one member of the Ohr/OsmC family, being arranged in highly variable configurations (Fig. 3). For instance, some fungi microorganisms present two or three ohr paralogues in their genomes (Fig. 3A and B), some located near to each other, in some cases the two gene are even neighbors (Fig. 3A), suggesting the occurrence of gene duplication events. Besides fungi, microorganisms that contain more than one gene of the Ohr/OsmC family are: M. fijiensis presenting one ohr and one ohr-like gene (Fig. 3C); Trichomonas vaginalis with four ohr-like genes (as also described by [22]) and D. discoideum AX4 containing one copy of osmC and one copy of an ohr-like gene (Fig. 3D). On the other hand, most of the other genomes encode only a single homolog of the ohr subfamily, as is the case of the moss P. patens (Fig. 3E).

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.