Figure 1.
Recycling of bacterial NAD catabolism products.
Reactions described in this study, numbered from 1 to 3, are catalyzed by: 1) NMN deamidase (PncC); 2) NMN adenylyltransferase of the NadM family; 3) ADPR pyrophosphatase. In several bacterial species PncC and NadM occur in fused forms with COG1058 ADPRP and Nudix ADPRP, respectively, as discussed in this work. Abbreviations: Nm, nicotinamide; NMN, nicotinamide mononucleotide; NaMN, nicotinate mononucleotide; NaAD, nicotinate adenine dinucleotide.
Figure 2.
Pyrophosphatase reactions catalyzed by bacterial MoeA and its eukaryotic ortholog (A) and ADP-ribose pyrophosphatase (B).
Both substrates share an adenosine group linked to two different moieties through the pyrophosphate bridge which is cleaved during the enzyme-catalyzed reaction.
Figure 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 37°C 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 µmol of product per min, under the specified conditions.
Figure 4.
Substrate specificity screening of AtCOG1058 and SoCOG1058/PncC pyrophosphatases.
The pyrophosphatase activity of the pure recombinant enzymes was assayed as described in “Materials and Methods”, in the presence of the listed compounds at 0.5 mM concentration each. Abbreviations: Ap3A, diadenosine triphosphate; Ap4A, diadenosine tetraphosphate; Ap5A, diadenosine pentaphosphate; NGD, nicotinamide guanine dinucleotide; NHD, nicotinamide hypoxanthine dinucleotide.
Figure 5.
Kinetic characterization of SoCOG1058/PncC and AtCOG1058 enzymes.
Plots of the initial velocities of the catalyzed reactions versus substrate concentrations. Kinetic parameters, calculated as described in Materials and Methods, are reported in the table.
Figure 6.
Phylogenetic distribution and domain composition of COG1058.
Schematic representation of bacterial (A), eukaryotic (B) and archaeal (C) species trees showing COG1058 genes mapping. Green circle designates the COG1058 gene; the FAD synthase gene is represented by a red circle; the fused COG1058/pncC gene is shown as a blue square. Numbers within squares represents the number of gene copies per genome.
Figure 7.
Schematic representation of the COG1058 phylogenetic tree (full version is in Fig. S1). The stand-alone COG1058 gene and the gene fused with FAD synthetase and pncC genes are depicted as green, red and blue circles, respectively. The Shewanella oneidensis and Agrobacterium tumefaciens COG1058 proteins, experimentally characterized in this work, are marked by red stars. Thermoplasma acidophilum COG1058 protein, whose 3D structure is available, is highlighted.
Figure 8.
Multiple sequence alignment of selected COG1058 proteins.
Multiple alignment of representative members of the COG1058 family (full version is available in Figure 2). Positions of residues conserved in all members of the family are highlighted at the top of the alignment in magenta. The residues highlighted in green are conserved in all proteins, with the exception of the plant subfamily. Residues are numbered according to the T. acidophilum protein. Proteins experimentally characterized in this work are marked by red stars. Residues mutated in the A.tumefaciens protein are marked with black asterisks.
Figure 9.
Structural comparison of Thermoplasma acidophilum COG1058 and E. coli MoeA enzymes.
A) Ribbon representation of superposed T. acidophilum COG1058 (blue) and E. coli MoeA (cyan) structures. The sulfate ion found in the COG1058 structure, likely indicative of the position of the active site, is shown as ball and stick; B) Superposed COG1058 and MoeA structures viewed from the top. The MoeA acidic residues predicted to be involved in catalysis and the two glycines of the conserved motif proposed to interact with the phosphate moiety of the MPT substrate are highlighted in orange, and their superposition to identical residues in the COG1058 structure is shown.