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

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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.

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Multiple sequence alignment of selected members from Ohr, OsmC and Ohr-like subfamilies. The sequences were aligned using L-INS-I algorithm of MAFFT [13]. For each subfamily, sequences from different bacteria phyla were aligned with selected Ohr eukaryotic sequences. (A) For Ohr, 4NOZ secondary structure from Burkholderia cenocepacia J2315 (Bc_Ohr) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected Ohr sequences of prokaryotes were: Rs_Ohr from Ralstonia solanacearum UW551, Sa_Ohr from Stigmatella aurantiaca, Pg_Ohr from Polymorphum gilvum, Pa_Ohr from Propionibacterium acnes, Cc_Ohr from Clostridium carboxidivorans, Sp_Ohr from Sphingobacterium paucimobilis, Ds_Ohr from Deinococcus swuensis, Mr_Ohr from Mastigocladopsis repens, Kr_Ohr from Ktedonobacter racemifer and Ma_Ohr from Mycoplasma alligatoris. The selected Ohr sequences of eukaryotes were: Pp_Ohr from Physcomitrella patens, Kf_Ohr from Klebsormidium flaccidum, Mf_Ohr from Mycosphaerella fijiensis CIRAD86, Pm_Ohr from Pseudocercospora musae, Fo_Ohr from Fusarium oxysporum f. sp. cubense race 4, Ai_Ohr from Aphanomyces invadans, Bd_Ohr from Batrachochytrium dendrobatidis JEL423, Cc_Ohr from Calocera cornea HHB12733 Cc_1, Rt_Ohr from Rhodotorula toruloides ATCC 20409, Me_Ohr from Mortierella elongata AG-77, Mv_Ohr from Mortierella verticillata NRRL 6337. (B) For OsmC, 1QLM secondary structure from Escherichia coli (Ec_OsmC) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected OsmC sequences of prokaryotes were: Vp_OsmC from Variovorax paradoxus, Ab_OsmC from Azospirillum brasilense, Bb_OsmC from Bdellovibrio bacteriovorus, Sa_OsmC from Streptomyces avermitilis, Fa_OsmC from Flavobacterium aquatile, Kr_OsmC from Ktedonobacter racemifer, Df_OsmC from Deinococcus frigens, Ll_OsmC from Lactococcus lactis, Fm_OsmC from Fischerella muscicola and Vs_OsmC from Verrucomicrobium spinosum. The selected OsmC sequences of eukaryotes were: Pp_OsmC from Polysphondylium pallidum PN500, Dp_OsmC from Dictyostelium purpureum, As_OsmC from Acytostelium subglobosum LB1 and Dd1_OsmC from Dictyostelium discoideum. (C) Selected Ohr-like sequences deposited in PDB database were aligned with selected Ohr-like members from eukaryotic counterparts. For Ohr-like, secondary structure 2PN2 from Psychrobacter arcticus 273-4 (Pa_Ohr_like) was used to guide the alignment. The selected OsmC sequences of prokaryotes were: Aa_Ohr_like from Aquifex aeolicus, Js_Ohr_like from Jannaschia sp., Ll_Ohr_like from Lactobacillus casei, Tm_Ohr_like from Thermotoga maritima, Ta_Ohr_like from Thermoplasma acidophilum. The selected Ohr-like sequences of eukaryotes were: Mp_Ohr_like from Micromonas pusilla CCMP1545, Ep_Ohr_like from Exaiptasia pallida, Tv_Ohr_like from Trichomonas vaginalis G3, To_Ohr_like from Thalassiosira oceanica, Ai_Ohr_like from Aphanomyces invadans, Mf_Ohr_like from Mychosphaerella fijiensis CIRAD86, Fo_Ohr_like from Fusarium oxysporum f. sp. cubense race 4, Cc_Ohr_like from Calocera cornea HHB12733, Rt_Ohr_like from Rhodotorula toruloides ATCC 204091 and Co_Ohr_like from Capsaspora owczarzaki ATCC 30864. (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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f0005: Multiple sequence alignment of selected members from Ohr, OsmC and Ohr-like subfamilies. The sequences were aligned using L-INS-I algorithm of MAFFT [13]. For each subfamily, sequences from different bacteria phyla were aligned with selected Ohr eukaryotic sequences. (A) For Ohr, 4NOZ secondary structure from Burkholderia cenocepacia J2315 (Bc_Ohr) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected Ohr sequences of prokaryotes were: Rs_Ohr from Ralstonia solanacearum UW551, Sa_Ohr from Stigmatella aurantiaca, Pg_Ohr from Polymorphum gilvum, Pa_Ohr from Propionibacterium acnes, Cc_Ohr from Clostridium carboxidivorans, Sp_Ohr from Sphingobacterium paucimobilis, Ds_Ohr from Deinococcus swuensis, Mr_Ohr from Mastigocladopsis repens, Kr_Ohr from Ktedonobacter racemifer and Ma_Ohr from Mycoplasma alligatoris. The selected Ohr sequences of eukaryotes were: Pp_Ohr from Physcomitrella patens, Kf_Ohr from Klebsormidium flaccidum, Mf_Ohr from Mycosphaerella fijiensis CIRAD86, Pm_Ohr from Pseudocercospora musae, Fo_Ohr from Fusarium oxysporum f. sp. cubense race 4, Ai_Ohr from Aphanomyces invadans, Bd_Ohr from Batrachochytrium dendrobatidis JEL423, Cc_Ohr from Calocera cornea HHB12733 Cc_1, Rt_Ohr from Rhodotorula toruloides ATCC 20409, Me_Ohr from Mortierella elongata AG-77, Mv_Ohr from Mortierella verticillata NRRL 6337. (B) For OsmC, 1QLM secondary structure from Escherichia coli (Ec_OsmC) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected OsmC sequences of prokaryotes were: Vp_OsmC from Variovorax paradoxus, Ab_OsmC from Azospirillum brasilense, Bb_OsmC from Bdellovibrio bacteriovorus, Sa_OsmC from Streptomyces avermitilis, Fa_OsmC from Flavobacterium aquatile, Kr_OsmC from Ktedonobacter racemifer, Df_OsmC from Deinococcus frigens, Ll_OsmC from Lactococcus lactis, Fm_OsmC from Fischerella muscicola and Vs_OsmC from Verrucomicrobium spinosum. The selected OsmC sequences of eukaryotes were: Pp_OsmC from Polysphondylium pallidum PN500, Dp_OsmC from Dictyostelium purpureum, As_OsmC from Acytostelium subglobosum LB1 and Dd1_OsmC from Dictyostelium discoideum. (C) Selected Ohr-like sequences deposited in PDB database were aligned with selected Ohr-like members from eukaryotic counterparts. For Ohr-like, secondary structure 2PN2 from Psychrobacter arcticus 273-4 (Pa_Ohr_like) was used to guide the alignment. The selected OsmC sequences of prokaryotes were: Aa_Ohr_like from Aquifex aeolicus, Js_Ohr_like from Jannaschia sp., Ll_Ohr_like from Lactobacillus casei, Tm_Ohr_like from Thermotoga maritima, Ta_Ohr_like from Thermoplasma acidophilum. The selected Ohr-like sequences of eukaryotes were: Mp_Ohr_like from Micromonas pusilla CCMP1545, Ep_Ohr_like from Exaiptasia pallida, Tv_Ohr_like from Trichomonas vaginalis G3, To_Ohr_like from Thalassiosira oceanica, Ai_Ohr_like from Aphanomyces invadans, Mf_Ohr_like from Mychosphaerella fijiensis CIRAD86, Fo_Ohr_like from Fusarium oxysporum f. sp. cubense race 4, Cc_Ohr_like from Calocera cornea HHB12733, Rt_Ohr_like from Rhodotorula toruloides ATCC 204091 and Co_Ohr_like from Capsaspora owczarzaki ATCC 30864. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Mentions: Ohr, OsmC and Ohr-like proteins share a conserved pair of catalytic cysteines separated by approximately 60 amino acid residues in the primary sequence (Fig. 1) that, therefore, represents a hallmark feature of Ohr/OsmC of family proteins. Two additional residues (an Arg and a Glu) required for the peroxidatic activity [2], [4], [6], [14] are both fully conserved in Ohr and OsmC subfamilies (Fig. 1A and B) but are absent in Ohr-like proteins (Fig. 1C). This conserved Glu residue is located at the same position in the primary sequences of Ohr and OsmC proteins, while the conserved Arg residue is located in the first loop between the 1st and 2nd β-sheets for Ohr proteins (Fig. 1A); and in the third loop between the 3rd β-sheet and the 1st α-helix for OsmC (Fig. 1B). Although the conserved Arg residue is present at different positions in the primary sequences of Ohr and OsmC enzymes, in the tertiary structures they occupy a similar orientation between the conserved Glu and Cp[1], [12].


Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi
Multiple sequence alignment of selected members from Ohr, OsmC and Ohr-like subfamilies. The sequences were aligned using L-INS-I algorithm of MAFFT [13]. For each subfamily, sequences from different bacteria phyla were aligned with selected Ohr eukaryotic sequences. (A) For Ohr, 4NOZ secondary structure from Burkholderia cenocepacia J2315 (Bc_Ohr) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected Ohr sequences of prokaryotes were: Rs_Ohr from Ralstonia solanacearum UW551, Sa_Ohr from Stigmatella aurantiaca, Pg_Ohr from Polymorphum gilvum, Pa_Ohr from Propionibacterium acnes, Cc_Ohr from Clostridium carboxidivorans, Sp_Ohr from Sphingobacterium paucimobilis, Ds_Ohr from Deinococcus swuensis, Mr_Ohr from Mastigocladopsis repens, Kr_Ohr from Ktedonobacter racemifer and Ma_Ohr from Mycoplasma alligatoris. The selected Ohr sequences of eukaryotes were: Pp_Ohr from Physcomitrella patens, Kf_Ohr from Klebsormidium flaccidum, Mf_Ohr from Mycosphaerella fijiensis CIRAD86, Pm_Ohr from Pseudocercospora musae, Fo_Ohr from Fusarium oxysporum f. sp. cubense race 4, Ai_Ohr from Aphanomyces invadans, Bd_Ohr from Batrachochytrium dendrobatidis JEL423, Cc_Ohr from Calocera cornea HHB12733 Cc_1, Rt_Ohr from Rhodotorula toruloides ATCC 20409, Me_Ohr from Mortierella elongata AG-77, Mv_Ohr from Mortierella verticillata NRRL 6337. (B) For OsmC, 1QLM secondary structure from Escherichia coli (Ec_OsmC) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected OsmC sequences of prokaryotes were: Vp_OsmC from Variovorax paradoxus, Ab_OsmC from Azospirillum brasilense, Bb_OsmC from Bdellovibrio bacteriovorus, Sa_OsmC from Streptomyces avermitilis, Fa_OsmC from Flavobacterium aquatile, Kr_OsmC from Ktedonobacter racemifer, Df_OsmC from Deinococcus frigens, Ll_OsmC from Lactococcus lactis, Fm_OsmC from Fischerella muscicola and Vs_OsmC from Verrucomicrobium spinosum. The selected OsmC sequences of eukaryotes were: Pp_OsmC from Polysphondylium pallidum PN500, Dp_OsmC from Dictyostelium purpureum, As_OsmC from Acytostelium subglobosum LB1 and Dd1_OsmC from Dictyostelium discoideum. (C) Selected Ohr-like sequences deposited in PDB database were aligned with selected Ohr-like members from eukaryotic counterparts. For Ohr-like, secondary structure 2PN2 from Psychrobacter arcticus 273-4 (Pa_Ohr_like) was used to guide the alignment. The selected OsmC sequences of prokaryotes were: Aa_Ohr_like from Aquifex aeolicus, Js_Ohr_like from Jannaschia sp., Ll_Ohr_like from Lactobacillus casei, Tm_Ohr_like from Thermotoga maritima, Ta_Ohr_like from Thermoplasma acidophilum. The selected Ohr-like sequences of eukaryotes were: Mp_Ohr_like from Micromonas pusilla CCMP1545, Ep_Ohr_like from Exaiptasia pallida, Tv_Ohr_like from Trichomonas vaginalis G3, To_Ohr_like from Thalassiosira oceanica, Ai_Ohr_like from Aphanomyces invadans, Mf_Ohr_like from Mychosphaerella fijiensis CIRAD86, Fo_Ohr_like from Fusarium oxysporum f. sp. cubense race 4, Cc_Ohr_like from Calocera cornea HHB12733, Rt_Ohr_like from Rhodotorula toruloides ATCC 204091 and Co_Ohr_like from Capsaspora owczarzaki ATCC 30864. (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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f0005: Multiple sequence alignment of selected members from Ohr, OsmC and Ohr-like subfamilies. The sequences were aligned using L-INS-I algorithm of MAFFT [13]. For each subfamily, sequences from different bacteria phyla were aligned with selected Ohr eukaryotic sequences. (A) For Ohr, 4NOZ secondary structure from Burkholderia cenocepacia J2315 (Bc_Ohr) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected Ohr sequences of prokaryotes were: Rs_Ohr from Ralstonia solanacearum UW551, Sa_Ohr from Stigmatella aurantiaca, Pg_Ohr from Polymorphum gilvum, Pa_Ohr from Propionibacterium acnes, Cc_Ohr from Clostridium carboxidivorans, Sp_Ohr from Sphingobacterium paucimobilis, Ds_Ohr from Deinococcus swuensis, Mr_Ohr from Mastigocladopsis repens, Kr_Ohr from Ktedonobacter racemifer and Ma_Ohr from Mycoplasma alligatoris. The selected Ohr sequences of eukaryotes were: Pp_Ohr from Physcomitrella patens, Kf_Ohr from Klebsormidium flaccidum, Mf_Ohr from Mycosphaerella fijiensis CIRAD86, Pm_Ohr from Pseudocercospora musae, Fo_Ohr from Fusarium oxysporum f. sp. cubense race 4, Ai_Ohr from Aphanomyces invadans, Bd_Ohr from Batrachochytrium dendrobatidis JEL423, Cc_Ohr from Calocera cornea HHB12733 Cc_1, Rt_Ohr from Rhodotorula toruloides ATCC 20409, Me_Ohr from Mortierella elongata AG-77, Mv_Ohr from Mortierella verticillata NRRL 6337. (B) For OsmC, 1QLM secondary structure from Escherichia coli (Ec_OsmC) was used to guide the alignment. Green and red arrows denote catalytic Arg and Glu. The selected OsmC sequences of prokaryotes were: Vp_OsmC from Variovorax paradoxus, Ab_OsmC from Azospirillum brasilense, Bb_OsmC from Bdellovibrio bacteriovorus, Sa_OsmC from Streptomyces avermitilis, Fa_OsmC from Flavobacterium aquatile, Kr_OsmC from Ktedonobacter racemifer, Df_OsmC from Deinococcus frigens, Ll_OsmC from Lactococcus lactis, Fm_OsmC from Fischerella muscicola and Vs_OsmC from Verrucomicrobium spinosum. The selected OsmC sequences of eukaryotes were: Pp_OsmC from Polysphondylium pallidum PN500, Dp_OsmC from Dictyostelium purpureum, As_OsmC from Acytostelium subglobosum LB1 and Dd1_OsmC from Dictyostelium discoideum. (C) Selected Ohr-like sequences deposited in PDB database were aligned with selected Ohr-like members from eukaryotic counterparts. For Ohr-like, secondary structure 2PN2 from Psychrobacter arcticus 273-4 (Pa_Ohr_like) was used to guide the alignment. The selected OsmC sequences of prokaryotes were: Aa_Ohr_like from Aquifex aeolicus, Js_Ohr_like from Jannaschia sp., Ll_Ohr_like from Lactobacillus casei, Tm_Ohr_like from Thermotoga maritima, Ta_Ohr_like from Thermoplasma acidophilum. The selected Ohr-like sequences of eukaryotes were: Mp_Ohr_like from Micromonas pusilla CCMP1545, Ep_Ohr_like from Exaiptasia pallida, Tv_Ohr_like from Trichomonas vaginalis G3, To_Ohr_like from Thalassiosira oceanica, Ai_Ohr_like from Aphanomyces invadans, Mf_Ohr_like from Mychosphaerella fijiensis CIRAD86, Fo_Ohr_like from Fusarium oxysporum f. sp. cubense race 4, Cc_Ohr_like from Calocera cornea HHB12733, Rt_Ohr_like from Rhodotorula toruloides ATCC 204091 and Co_Ohr_like from Capsaspora owczarzaki ATCC 30864. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Mentions: Ohr, OsmC and Ohr-like proteins share a conserved pair of catalytic cysteines separated by approximately 60 amino acid residues in the primary sequence (Fig. 1) that, therefore, represents a hallmark feature of Ohr/OsmC of family proteins. Two additional residues (an Arg and a Glu) required for the peroxidatic activity [2], [4], [6], [14] are both fully conserved in Ohr and OsmC subfamilies (Fig. 1A and B) but are absent in Ohr-like proteins (Fig. 1C). This conserved Glu residue is located at the same position in the primary sequences of Ohr and OsmC proteins, while the conserved Arg residue is located in the first loop between the 1st and 2nd β-sheets for Ohr proteins (Fig. 1A); and in the third loop between the 3rd β-sheet and the 1st α-helix for OsmC (Fig. 1B). Although the conserved Arg residue is present at different positions in the primary sequences of Ohr and OsmC enzymes, in the tertiary structures they occupy a similar orientation between the conserved Glu and Cp[1], [12].

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.


Related in: MedlinePlus