Kinetic parameters of wild-type OiNIC and its mutants.

a<p>Reactions were carried out following the standard spectrophotometric method.</p>b<p>Reactions were analyzed by standard HPLC method.</p

Structural alignment of modelled OiNIC and crystallized <i>Mycobacterium tuberculosis</i> MtPncA.

<p>A) Monomer of OiNIC is represented in red and monomer of MtPncA in blue. Fe²⁺ and Zn²⁺ are represented as blue and red spheres, respectively. The arrow represents the 51–71 loop of MtPncA. NAM is colored in green. B) Residues interacting with the metal ion. C) Residues forming the active site cavity and interacting with NAM.</p

Phylogenetic distribution of bacterial nicotinamidases.

<p>For reasons of clarity, branches are shown compressed as triangles. The scale bar at the lower left indicates the rate of amino acids substitutions. The triangle base corresponds to the number of compressed sequences involved, which is also shown in parentheses. The triangle height corresponds to evolutionary distance. The bacterial nicotinamidase sequences used in this study (see text for details) are phylogenetically divided into 4 groups, in which biochemically characterized nicotinamidases are positioned according with its activity towards nicotinamide and/or pyrazinamide. <i>E. coli</i> isochorismatase was used as outgroup. The phylogenetic tree was obtained using MEGA 5.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056727#pone.0056727-Tamura1" target="_blank">[53]</a>.</p

Plot of the different Pfam domain architectures found for PncC enzymes using ArchSchema.

<p>Green rectangles represent the CinA domain (Pfam ID: PF02464). Red rectangles represent the MocF domain (Pfam ID: PF00994). Other colored rectangles and squares represent other Pfam domains. Labels represent UniProt codes of enzymes belonging to each architecture. </p

Nicotinamidase distribution (A) in biology and (B) among bacteria.

<p>Nicotinamidase distribution (A) in biology and (B) among bacteria.</p

Effect of pH and temperature on OiNIC activity and stability.

<p>A) pH profile for OiNIC determined by HPLC. The assay conditions at 37°C were 1 mM nicotinamide, 0.67 µg of OiNIC. The buffers (100 mM) used were sodium acetate pH 4.0–6.0, sodium phosphate pH 7.0–8.0, Tris-HCl pH 9.0 and glycine pH 10.0. B) Temperature profile. Assay conditions were the same as above at pH 7.3 at different temperatures from 15 to 65°C. C) pH stability. OiNIC was incubated at 37°C at pH 5 (•), pH 6 (▪), pH 7.3 (Δ), pH 8 (▾), pH 9 (◊) and pH 10(○). Buffer compositions were the same as above. Residual activity was measured spectrophotometrically under the standard reaction at 37°C. D) Temperature stability. OiNIC was incubated at 4 (•), 20 (▪), 37 (Δ), 45 (▾) and 55°C (◊) in 100 mM sodium phosphate buffer pH 7.3. Residual activity was measured spectrophotometrically using the standard reaction medium.</p

Multiple sequence alignment for <i>O. iheyensis</i> nicotinamidase (OiNIC) and related nicotinamidases.

<p>ESPript outputs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056727#pone.0056727-Gouet1" target="_blank">[48]</a> obtained with the crystallized nicotinamidase sequences retrieved from Uniprot database and later aligned with CLUSTAL-W <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056727#pone.0056727-Thompson1" target="_blank">[47]</a>. Sequences are grouped according to similarity. PDB codes were 3O90 for <i>S. pneumoniae</i> nicotinamidase (SpNIC), 1ILW for <i>P. horikoshii</i> nicotinamidase (PhPncA), 3PL1 for <i>M. tuberculosis</i> nicotinamidase (MtPncA), 2WT9 for <i>A. baumanii</i> nicotinamidase (AbPncA), 2H0R for <i>S. cerevisiae</i> nicotinamidase and 2R3J for <i>Leishmania infantum</i> nicotinamidase. Residues strictly conserved across nicotinamidase enzymes have a dark background. Symbols above blocks of sequences represent the secondary structure, springs represent helices and arrows represent β-strands. The residues forming the active site are indicated by triangles. Residues involved in the coordination of the metal ion are indicated by stars and residues forming the <i>cis</i>-peptide bond are indicated by diamonds.</p

Effect of pH and temperature on OiPncC.

<p><b>A</b>) Effect of pH on OiPncC activity measured by the HPLC Assay. The buffers used were 50 mM sodium acetate (pH 5.0), 50 mM potassium phosphate buffer (pH 6.0-7.4) and glycine-NaOH (pH 8.5-10.0). <b>B</b>) pH-stability. Aliquots of enzyme incubated at different pHs were removed and relative activity was measured using the enzyme-coupled assay at different times. The buffers used (50mM) were sodium acetate pH 5.0 (●), potassium phosphate pH 6.5 (■), pH 7.0 (Δ), pH 8.0 (▲), Tris-HCl pH 9.0 (♦), glycine pH 10 (□) and pH 10.5 (◊). <b>C</b>) Effect of temperature on OiPncC activity measured by the HPLC assay. <b>D</b>) Thermostability assay. Aliquots of enzyme incubated at different temperatures [4 °C (●), 20 °C (■), 37 °C (Δ), 45 °C (▲), 50 °C (♦) and 60 °C (◊)] were removed at different times and relative activity was measured using the enzyme-coupled assay. Standard assay conditions were used in all cases.</p

Distribution analysis of NMN deamidases.

<p>The figure shows a representative Tree of Life based on 16S rRNA. Species of Kingdom Eukarya are coloured red, those of Kingdom Archaea are coloured yellow and those of Kingdom Bacteria are coloured blue. The box next to each species represent the absence of OiPncC (purple), the presence of the functional one-domain enzyme (green) or the presence of a functional two-domain enzyme (orange) in the organism. The image was generated with iTOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082705#B31" target="_blank">31</a>]. .</p

Structural analysis of MocF domain.

<p>A) Surface (subunit A) and ribbon (subunit B) representation of the dimeric <i>Thermoplasma acidophilum</i> CinA protein (PDB code: 3KBQ); conserved blocks forming the binding site are colored, and its consensus sequence shown as generated by WebLogo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082705#B34" target="_blank">34</a>]. A Molybdenum cofactor molecule (Moco) in the proposed binding site is shown in ball and stick representation. B) Detailed view of the amino acids involved in the interaction between 3KBQ and Moco rendered by Chimera [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082705#B25" target="_blank">25</a>]. C) An ADPr molecule in the proposed binding site (subunit A) is shown in ball and stick representation. D) Detailed view of the amino acids involved in the interaction between 3KBQ and ADPr.</p