Rmatics Tools and ResourcesThe Title Loaded From File Title Loaded From File COG1058 protein sequences in available complete genomes were taken from The SEED comparative genomics database [23]. Due to the large number of sequences 10781694 retrieved, a special procedure had to be used for the construction of multiple sequence alignment: i) an approximate phylogenetic tree was built by the FastTree tool [24]; ii) all sequences were divided into fifteenCOG1058 Is a Novel Pyrophosphatase FamilyCOG1058 Is a Novel Pyrophosphatase FamilyFigure 3. Characterization of ADP-ribose hydrolysis by recombinant A. tumefaciens COG1058 enzyme. Enzymatic assays were performed in the presence of 0.5 mM ADPR and 10 ng of pure protein. Reaction mixtures were incubated for 10 min at 37uC in: A) 100 mM HEPES/KOH, pH 7.5, in the presence of different divalent cations at 1 mM concentration (all ions were added as chloride salts); B) 100 mM HEPES/KOH, pH 7.5, with different concentrations of MgCl2 or CoCl2; C) 100 mM TRIS/HCl buffer, pH 7.5, 1 mM Co+2, in the presence of 10 mM and 100 mM of the indicated monovalent cations (added as chloride salts); D) 100 mM TRIS/HCl, pH 7.5 and 100 mM HEPES/KOH, pH 7.5, 1 mM Co+2, in the presence of different K+ concentrations (K+ ions were added as KCl); E) different buffer species at 100 mM concentration, pH 7.5, 1 mM Co+2, 0.1 M K+; F) 100 mM BIS-TRIS buffer at varying pH values, 1 mM Co+2, 0.1 M K+. One Unit of enzyme activity represents the amount of enzyme catalyzing the formation of 1 mmol of product per min, under the specified conditions. doi:10.1371/journal.pone.0065595.gclusters corresponding to the separate branches of the tree; iii) multiple alignment of sequences belonging to the same cluster was obtained using Clustal Omega [25]; iv) poorly aligned regions were cut from the cluster alignments; v) the final alignment was constructed using the profile-to-profile alignment option of the Clustal Omega algorithm. The phylogenetic tree was built by RAxML [26]. The species tree was taken from the Superfamily database [27]. Visualization of protein three-dimensional structures and structure comparison were performed using Chimera [28]. Multiple sequence alignment figures were prepared using TeXshade [29]. Genome context analysis was performed in The SEED environment.Results Bacterial Members of the COG1058 Family are Endowed with ADP-ribose Pyrophosphatase ActivityBoth Shewanella oneidensis (So) COG1058/PncC protein, in which the COG1058 domain is fused with the NMN deamidase (PncC) domain, and A. tumefaciens (At) COG1058 protein (gi 159184889), which comprises only the COG1058 domain, were assayed for the ADPRP activity. Both proteins were found to possess such activity in HEPES/KOH buffer, pH 7.5, 1.0 mM Mg+2. The ADPRP activity of the At enzyme was further characterized in order todetermine the optimal conditions for the reaction. Catalysis resulted to be metal-dependent (Figure 3A). Among the tested divalent cations, Co+2 was the most effective in supporting the enzyme activity, with Ni+2, Mg+2 and Mn+2 being about seven-fold less efficient; 1 mM Ca+2, Cu+2 and Zn+2 did not sustain the activity at all (Figure 3A). Mg+2 and Co+2 titration experiments showed that the enzyme-catalyzed ADPR hydrolysis was 1676428 optimal at 0.5 mM Co+2 (Figure 3B), while 10 mM Mg+2 was needed to reach the maximum activity, corresponding to about 30 of the optimal Co+2-dependent activity (not shown). We found that the presence in the reaction mixtures of a monovalent cation was also essential for t.Rmatics Tools and ResourcesThe COG1058 protein sequences in available complete genomes were taken from The SEED comparative genomics database [23]. Due to the large number of sequences 10781694 retrieved, a special procedure had to be used for the construction of multiple sequence alignment: i) an approximate phylogenetic tree was built by the FastTree tool [24]; ii) all sequences were divided into fifteenCOG1058 Is a Novel Pyrophosphatase FamilyCOG1058 Is a Novel Pyrophosphatase FamilyFigure 3. Characterization of ADP-ribose hydrolysis by recombinant A. tumefaciens COG1058 enzyme. Enzymatic assays were performed in the presence of 0.5 mM ADPR and 10 ng of pure protein. Reaction mixtures were incubated for 10 min at 37uC in: A) 100 mM HEPES/KOH, pH 7.5, in the presence of different divalent cations at 1 mM concentration (all ions were added as chloride salts); B) 100 mM HEPES/KOH, pH 7.5, with different concentrations of MgCl2 or CoCl2; C) 100 mM TRIS/HCl buffer, pH 7.5, 1 mM Co+2, in the presence of 10 mM and 100 mM of the indicated monovalent cations (added as chloride salts); D) 100 mM TRIS/HCl, pH 7.5 and 100 mM HEPES/KOH, pH 7.5, 1 mM Co+2, in the presence of different K+ concentrations (K+ ions were added as KCl); E) different buffer species at 100 mM concentration, pH 7.5, 1 mM Co+2, 0.1 M K+; F) 100 mM BIS-TRIS buffer at varying pH values, 1 mM Co+2, 0.1 M K+. One Unit of enzyme activity represents the amount of enzyme catalyzing the formation of 1 mmol of product per min, under the specified conditions. doi:10.1371/journal.pone.0065595.gclusters corresponding to the separate branches of the tree; iii) multiple alignment of sequences belonging to the same cluster was obtained using Clustal Omega [25]; iv) poorly aligned regions were cut from the cluster alignments; v) the final alignment was constructed using the profile-to-profile alignment option of the Clustal Omega algorithm. The phylogenetic tree was built by RAxML [26]. The species tree was taken from the Superfamily database [27]. Visualization of protein three-dimensional structures and structure comparison were performed using Chimera [28]. Multiple sequence alignment figures were prepared using TeXshade [29]. Genome context analysis was performed in The SEED environment.Results Bacterial Members of the COG1058 Family are Endowed with ADP-ribose Pyrophosphatase ActivityBoth Shewanella oneidensis (So) COG1058/PncC protein, in which the COG1058 domain is fused with the NMN deamidase (PncC) domain, and A. tumefaciens (At) COG1058 protein (gi 159184889), which comprises only the COG1058 domain, were assayed for the ADPRP activity. Both proteins were found to possess such activity in HEPES/KOH buffer, pH 7.5, 1.0 mM Mg+2. The ADPRP activity of the At enzyme was further characterized in order todetermine the optimal conditions for the reaction. Catalysis resulted to be metal-dependent (Figure 3A). Among the tested divalent cations, Co+2 was the most effective in supporting the enzyme activity, with Ni+2, Mg+2 and Mn+2 being about seven-fold less efficient; 1 mM Ca+2, Cu+2 and Zn+2 did not sustain the activity at all (Figure 3A). Mg+2 and Co+2 titration experiments showed that the enzyme-catalyzed ADPR hydrolysis was 1676428 optimal at 0.5 mM Co+2 (Figure 3B), while 10 mM Mg+2 was needed to reach the maximum activity, corresponding to about 30 of the optimal Co+2-dependent activity (not shown). We found that the presence in the reaction mixtures of a monovalent cation was also essential for t.
