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In Silico/In Vivo Insights into the Functional and Evolutionary Pathway of Pseudomonas aeruginosa Oleate-Diol Synthase. Discovery of a New Bacterial Di-Heme Cytochrome C Peroxidase Subfamily.

Estupiñán M, Álvarez-García D, Barril X, Diaz P, Manresa A - PLoS ONE (2015)

Bottom Line: From the 3D model obtained, presence of significant amino acid variations in the predicted heme-environment was found.These observations and the constructed phylogenetic tree of the two proteins, allow the proposal of an evolutionary pathway for P. aeruginosa oleate-diol synthase operon.Taking together the in silico and in vivo results obtained we conclude that enzymes PA2077 and PA2078 are the first described members of a new subfamily of bacterial peroxidases, designated as Fatty acid-di-heme Cytochrome c peroxidases (FadCcp).

View Article: PubMed Central - PubMed

Affiliation: Unitat de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, University of Barcelona, Barcelona, Spain; Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain.

ABSTRACT
As previously reported, P. aeruginosa genes PA2077 and PA2078 code for 10S-DOX (10S-Dioxygenase) and 7,10-DS (7,10-Diol Synthase) enzymes involved in long-chain fatty acid oxygenation through the recently described oleate-diol synthase pathway. Analysis of the amino acid sequence of both enzymes revealed the presence of two heme-binding motifs (CXXCH) on each protein. Phylogenetic analysis showed the relation of both proteins to bacterial di-heme cytochrome c peroxidases (Ccps), similar to Xanthomonas sp. 35Y rubber oxidase RoxA. Structural homology modelling of PA2077 and PA2078 was achieved using RoxA (pdb 4b2n) as a template. From the 3D model obtained, presence of significant amino acid variations in the predicted heme-environment was found. Moreover, the presence of palindromic repeats located in enzyme-coding regions, acting as protein evolution elements, is reported here for the first time in P. aeruginosa genome. These observations and the constructed phylogenetic tree of the two proteins, allow the proposal of an evolutionary pathway for P. aeruginosa oleate-diol synthase operon. Taking together the in silico and in vivo results obtained we conclude that enzymes PA2077 and PA2078 are the first described members of a new subfamily of bacterial peroxidases, designated as Fatty acid-di-heme Cytochrome c peroxidases (FadCcp).

No MeSH data available.


Products released from oleic acid by wild type PA2077 (A) and mutant PA2077 H130Q (B).Hydroperoxide 10-H(P)OME (RT = 11) could only be detected for wild type PA2077, whereas no conversion of oleic acid occurred when both mutants, PA2077 H130Q and PA2077 H375Q, were assayed.
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pone.0131462.g005: Products released from oleic acid by wild type PA2077 (A) and mutant PA2077 H130Q (B).Hydroperoxide 10-H(P)OME (RT = 11) could only be detected for wild type PA2077, whereas no conversion of oleic acid occurred when both mutants, PA2077 H130Q and PA2077 H375Q, were assayed.

Mentions: Analysis of the nucleotide sequence of ORFs PA2077 and PA2078 revealed the presence of short inverted repeats (IR) of 19 and 22 amino acids, inserted at positions 716 and 870, respectively (Table 1). On the contrary, no IR insertions were found in roxA nucleotide sequence. In silico removal of the IR insertions from the original nucleotide sequence of PA2077 and PA2078 produced a frame-readout from amino acid 240 in PA2078, which would result in loss of the C-terminal heme-binding motif CAACH, supporting the previous idea that PA2078 might not use the distal heme-binding site for activity, acting as a mono-heme enzyme. On the other hand, when the same procedure was applied to PA2077, the two CXXCH motifs were maintained, suggesting that they are required for activity. To prove this hypothesis, mutants of PA2077 and PA2078 at the proximal and distal heme motifs (Fig 1) were experimentally constructed and tested for activity on their corresponding substrates. Mutants at either position H130 or H375 (residue numbering with signal peptide) in protein PA2077 failed to convert oleic acid into 10-H(P)OME (Fig 5), definitely showing that both histidines (the two hemes) are required by PA2077 to display activity. However, mutants H130Q, H365Q and C518S constructed for PA2078 (numbering includes signal peptide) produced the same pattern as wild type PA2078 when assayed for conversion of 10-H(P)OME into 7,10-DiHOME (S3 Fig). These results indicate that neither the proximal or distal heme groups, nor the cysteine putatively related to P450 enzymes are involved in the catalytic activity of PA2078. Thus, despite being a di-heme enzyme, none of the heme groups of PA2078 seems to be involved in 10-H(P)OME conversion. Further mutagenesis approaches are required to completely elucidate the catalytic environment of PA2078, which must be located elsewhere in the protein sequence, out from the heme region.


In Silico/In Vivo Insights into the Functional and Evolutionary Pathway of Pseudomonas aeruginosa Oleate-Diol Synthase. Discovery of a New Bacterial Di-Heme Cytochrome C Peroxidase Subfamily.

Estupiñán M, Álvarez-García D, Barril X, Diaz P, Manresa A - PLoS ONE (2015)

Products released from oleic acid by wild type PA2077 (A) and mutant PA2077 H130Q (B).Hydroperoxide 10-H(P)OME (RT = 11) could only be detected for wild type PA2077, whereas no conversion of oleic acid occurred when both mutants, PA2077 H130Q and PA2077 H375Q, were assayed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496055&req=5

pone.0131462.g005: Products released from oleic acid by wild type PA2077 (A) and mutant PA2077 H130Q (B).Hydroperoxide 10-H(P)OME (RT = 11) could only be detected for wild type PA2077, whereas no conversion of oleic acid occurred when both mutants, PA2077 H130Q and PA2077 H375Q, were assayed.
Mentions: Analysis of the nucleotide sequence of ORFs PA2077 and PA2078 revealed the presence of short inverted repeats (IR) of 19 and 22 amino acids, inserted at positions 716 and 870, respectively (Table 1). On the contrary, no IR insertions were found in roxA nucleotide sequence. In silico removal of the IR insertions from the original nucleotide sequence of PA2077 and PA2078 produced a frame-readout from amino acid 240 in PA2078, which would result in loss of the C-terminal heme-binding motif CAACH, supporting the previous idea that PA2078 might not use the distal heme-binding site for activity, acting as a mono-heme enzyme. On the other hand, when the same procedure was applied to PA2077, the two CXXCH motifs were maintained, suggesting that they are required for activity. To prove this hypothesis, mutants of PA2077 and PA2078 at the proximal and distal heme motifs (Fig 1) were experimentally constructed and tested for activity on their corresponding substrates. Mutants at either position H130 or H375 (residue numbering with signal peptide) in protein PA2077 failed to convert oleic acid into 10-H(P)OME (Fig 5), definitely showing that both histidines (the two hemes) are required by PA2077 to display activity. However, mutants H130Q, H365Q and C518S constructed for PA2078 (numbering includes signal peptide) produced the same pattern as wild type PA2078 when assayed for conversion of 10-H(P)OME into 7,10-DiHOME (S3 Fig). These results indicate that neither the proximal or distal heme groups, nor the cysteine putatively related to P450 enzymes are involved in the catalytic activity of PA2078. Thus, despite being a di-heme enzyme, none of the heme groups of PA2078 seems to be involved in 10-H(P)OME conversion. Further mutagenesis approaches are required to completely elucidate the catalytic environment of PA2078, which must be located elsewhere in the protein sequence, out from the heme region.

Bottom Line: From the 3D model obtained, presence of significant amino acid variations in the predicted heme-environment was found.These observations and the constructed phylogenetic tree of the two proteins, allow the proposal of an evolutionary pathway for P. aeruginosa oleate-diol synthase operon.Taking together the in silico and in vivo results obtained we conclude that enzymes PA2077 and PA2078 are the first described members of a new subfamily of bacterial peroxidases, designated as Fatty acid-di-heme Cytochrome c peroxidases (FadCcp).

View Article: PubMed Central - PubMed

Affiliation: Unitat de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, University of Barcelona, Barcelona, Spain; Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain.

ABSTRACT
As previously reported, P. aeruginosa genes PA2077 and PA2078 code for 10S-DOX (10S-Dioxygenase) and 7,10-DS (7,10-Diol Synthase) enzymes involved in long-chain fatty acid oxygenation through the recently described oleate-diol synthase pathway. Analysis of the amino acid sequence of both enzymes revealed the presence of two heme-binding motifs (CXXCH) on each protein. Phylogenetic analysis showed the relation of both proteins to bacterial di-heme cytochrome c peroxidases (Ccps), similar to Xanthomonas sp. 35Y rubber oxidase RoxA. Structural homology modelling of PA2077 and PA2078 was achieved using RoxA (pdb 4b2n) as a template. From the 3D model obtained, presence of significant amino acid variations in the predicted heme-environment was found. Moreover, the presence of palindromic repeats located in enzyme-coding regions, acting as protein evolution elements, is reported here for the first time in P. aeruginosa genome. These observations and the constructed phylogenetic tree of the two proteins, allow the proposal of an evolutionary pathway for P. aeruginosa oleate-diol synthase operon. Taking together the in silico and in vivo results obtained we conclude that enzymes PA2077 and PA2078 are the first described members of a new subfamily of bacterial peroxidases, designated as Fatty acid-di-heme Cytochrome c peroxidases (FadCcp).

No MeSH data available.