Kinetic characterization of <i>So</i>COG1058/PncC and <i>At</i>COG1058 enzymes.

<p>Plots of the initial velocities of the catalyzed reactions <i>versus</i> substrate concentrations. Kinetic parameters, calculated as described in Materials and Methods, are reported in the table.</p

Multiple sequence alignment of selected COG1058 proteins.

<p>Multiple alignment of representative members of the COG1058 family (full version is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065595#pone-0065595-g002" target="_blank">Figure 2</a>). 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 <i>T. acidophilum</i> protein. Proteins experimentally characterized in this work are marked by red stars. Residues mutated in the <i>A.tumefaciens</i> protein are marked with black asterisks.</p

Phylogenetic distribution and domain composition of COG1058.

<p>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.</p

Substrate specificity screening of <i>At</i>COG1058 and <i>So</i>COG1058/PncC pyrophosphatases.

<p>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.</p

Genomics-Guided Analysis of NAD Recycling Yields Functional Elucidation of COG1058 as a New Family of Pyrophosphatases

<div><p>We have recently identified the enzyme NMN deamidase (PncC), which plays a key role in the regeneration of NAD in bacteria by recycling back to the coenzyme the pyridine by-products of its non redox consumption. In several bacterial species, PncC is fused to a COG1058 domain of unknown function, highly conserved and widely distributed in all living organisms. Here, we demonstrate that the PncC-fused domain is endowed with a novel Co⁺²- and K⁺-dependent ADP-ribose pyrophosphatase activity, and discuss the functional connection of such an activity with NAD recycling. An in-depth phylogenetic analysis of the COG1058 domain evidenced that in most bacterial species it is fused to PncC, while in α- and some δ-proteobacteria, as well as in archaea and fungi, it occurs as a stand-alone protein. Notably, in mammals and plants it is fused to FAD synthase. We extended the enzymatic characterization to a representative bacterial single-domain protein, which resulted to be a more versatile ADP-ribose pyrophosphatase, active also towards diadenosine 5′-diphosphate and FAD. Multiple sequence alignment analysis, and superposition of the available three-dimensional structure of an archaeal COG1058 member with the structure of the enzyme MoeA of the molybdenum cofactor biosynthesis, allowed identification of residues likely involved in catalysis. Their role has been confirmed by site-directed mutagenesis.</p></div

Activity profiling of mNMNAT isozymes in brain and liver from wild-type mouse and Wld^S mutant.

<p>Tissues were collected from 1 month old animals. The isozymes activities, measured by the described discrimination assay procedure, are color-coded as indicated (see graph legend). Tissue contents of total NMNAT activity and NAD, respectively determined under reference assay condition “A” and by HPLC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053271#s2" target="_blank">Methods</a>), are also shown. Each bar represents the average (+/− SD) from at least a triplicate experiment.</p

Characterization of ADP-ribose hydrolysis by recombinant <i>A. tumefaciens</i> COG1058 enzyme.

<p>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.</p

Bacterial overexpression and stability of recombinant isozyme preparations.

<p>Panel A) SDS-PAGE analysis of affinity purified His-tagged recombinant proteins. Gel lanes were loaded with standard protein markers of indicated mass values and 3–4 µg each of Wld^S (1), mNMNAT1 (2), mNMNAT2 (3), or mNMNAT3 (4) final enzyme preparation. ProtParam predicted masses of recombinant species are 44001 (1), 34518 (2), 36668 (3), and 29866 (4) daltons. Panel B) Stability of each preparation at 4°C and −20°C in a buffer solution composed by 50 mM HEPES/KOH, pH 7.5, 20% glycerol, and 1 mM TCEP.</p

Phylogenetic tree of COG1058.

<p>Schematic representation of the COG1058 phylogenetic tree (full version is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065595#pone.0065595.s001" target="_blank">Fig. S1</a>). 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 <i>Shewanella oneidensis</i> and <i>Agrobacterium tumefaciens</i> COG1058 proteins, experimentally characterized in this work, are marked by red stars. <i>Thermoplasma acidophilum</i> COG1058 protein, whose 3D structure is available, is highlighted.</p

In vitro discrimination of mNMNAT activities (mU).

<p>Table shows data from a typical discrimination experiment where the three recombinant mouse isozymes have been assayed both separately and after mixing in a reconstituted sample. The four assay conditions A, B, C, and D are described both in the text and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053271#s2" target="_blank">Methods</a>. On the right, respectively, the activity values resulting after matrix calculation and the percent errors with respect to the measured values under reference condition “A”. (^b) values in brackets are the sum of individual isozyme activities.</p