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

Phylogenetic analysis of NMN deamidases.

<p>The structures behind each organism name represent domain composition of the enzyme: MocF domain (dark blue); functional CinA domain (green); non-functional CinA domain (red); Eukaryotic PncCs (yellow). Special domains with only MocF domain are shown in light blue. C-terminal section of the protein is the outer part of the domain representation. Branch colours represent the same phylum as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082705#pone-0082705-g004" target="_blank">Figure 4</a>. The tree was built using Archaeopteryx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082705#B29" target="_blank">29</a>], bootstrap values were obtained after 1000 generations. The arrow and the star indicate the position of OiPncC and <i>S. oneidensis</i> PncC, respectively. Other symbols are: <i>E. coli</i> YFAY (▲), <i>E. coli</i> YDEJ (●), <i>E. coli</i> YGAD (○), <i>T. acidophilum</i> CinA (◊) and <i>A. tumefaciens</i> CinA (♦).</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

Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from <i>Agrobacterium tumefaciens</i> showing high thermal stability and catalytic efficiency

<div><p>NAD⁺ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organismal homeostasis. Among the enzymes involved in its recycling, nicotinamide mononucleotide (NMN) deamidase is one of the key players in the bacterial pyridine nucleotide cycle, where it catalyzes the conversion of NMN into nicotinic acid mononucleotide (NaMN), which is later converted to NAD⁺ in the Preiss-Handler pathway. The biochemical characteristics of bacterial NMN deamidases have been poorly studied, although they have been investigated in some firmicutes, gamma-proteobacteria and actinobacteria. In this study, we present the first characterization of an NMN deamidase from an alphaproteobacterium, <i>Agrobacterium tumefaciens</i> (AtCinA). The enzyme was active over a broad pH range, with an optimum at pH 7.5. Moreover, the enzyme was quite stable at neutral pH, maintaining 55% of its activity after 14 days. Surprisingly, AtCinA showed the highest optimal (80°C) and melting (85°C) temperatures described for an NMN deamidase. The above described characteristics, together with its high catalytic efficiency, make AtCinA a promising biocatalyst for the production of pure NaMN. In addition, six mutants (C32A, S48A, Y58F, Y58A, T105A and R145A) were designed to study their involvement in substrate binding, and two (S31A and K63A) to determine their contribution to the catalysis. However, only four mutants (C32A, S48A Y58F and T105A) showed activity, although with reduced catalytic efficiency. These results, combined with a thermal and structural analysis, reinforce the Ser/Lys catalytic dyad mechanism as the most plausible among those proposed.</p></div

Pyridine nucleotide cycle and NAD⁺ biosynthetic routes.

<p>The routes known to be functional across diverse bacterial species are shown by solid lines. Preiss-Handler pathway is shadowed. Dashed and hollow arrows relate to uptake and <i>de </i><i>novo</i> NaMN synthesis, respectively. Blue arrow corresponds to nicotinamidase activity (PncA), Red arrow corresponds to nicotinamide mononucleotide deamidase activity (PncC) and green arrow corresponds to NAD⁺-dependent DNA ligase. Enzymes are indicated as the acronym used to identify the corresponding gene locus: NadA, quinolinate synthetase; NadB, L-aspartate oxidase; NadC, quinolinate phosphoribosyl transferase; NadD, NaMN adenylyltransferase; NadE, NAD synthetase; NadM, NMN adenyltransferase; NadR^C, NmR kinase; NadR^N, NMN adenylyltransferase; NadV, Nm phosphoribosyltransferase; PncA, Nam deamidase; PncB, Na phosphoribosyltransferase; PncC, nicotinamide mononucleotide deamidase; PncU, nucleoside permease. The gene name of the enzymes existing in <i>O. iheyensis</i> is highlighted in brown. Abbreviations: NAD, nicotinamide adenine dinucleotide; IA, α-iminosuccinate; Qa, quinolonic acid; Asp, aspartate; Trp, tryptophan; NAM, nicotinamide; NA: nicotinic acid; NMN, nicotinic acid mononucleotide; NaAD, nicotinic acid adenine dinucleotide; NmR, nicotinamide riboside; DNA, deoxyribonucleic acid.</p

Structural analysis of CinA domain.

<p>A) Surface and ribbon representation of the <i>Agrobacterium tumefaciens</i> CinA dimer (PDB code: 2A9S); conserved blocks forming the binding site are colored and its consensus sequence are shown. A NMN molecule in the proposed binding site is shown in ball and stick representation. B) Detailed view of the amino acids involved in the interaction between 2A9S and NMN. C) Logo representations of the multiple alignments of the conserved blocks of CinA domain in representative active and inactive PncCs. The key residues involved in the catalytic process are marked with a red triangle.</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