Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.

A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld(S)) mouse. Wld(S) is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld(S)

Biosintesi della vitamina B3: studi strutturali e funzionali di isoenzimi nicotinamide mononucleotide adenililtrasferasi coinvolti nella protezione dalla neurodegenerazione

La Nicotinamide MonoNucleotide AdenililTrasferasi (NMNAT) è l’enzima chiave per la biosintesi del NAD+, sia nella via de novo che in quelle di recupero (salvage pathways). Nelle vie di recupero, l’NMNAT consente alla cellula di rigenerare il coenzima dalla vitamina B3 (niacina) o PP (Pellagra Preventing) assunta attraverso fonti esogene o ottenuta metabolicamente dalla degradazione intracellulare del dinucleotide stesso. Catalizzando il trasferimento dell’adenilato dall’ATP all’NMN, la forma mononucleotidica della vitamina B3, l’enzima porta alla formazione di PPi e NAD+. Il NAD+ è coinvolto nella cellula in vitali funzioni redox e non redox e la sua carenza è associata ad una vasta gamma di patologie nell’uomo, tra cui malattie neurodenegerative. È stato scoperto che la proteina chimerica WldS, proveniente da una mutazione spontanea sul cromosoma 4 nel ceppo murino C57BL/6J, è in grado di ritardare la degenerazione assonale. La porzione enzimaticamente attiva della proteina chimerica è l’omologo murino dell’enzima umano NMNAT1, una delle 3 isoforme note di NMNAT nei mammiferi che catalizzano la biosintesi del NAD+ in diversi distretti cellulari. Diversi lavori pubblicati hanno mostrato la capacità delle 3 isoforme di conferire protezione in varia misura, anche se non concordano su quale sia l’isoforma responsabile della protezione dalla neurodegenerazione. Per chiarire i meccanismi molecolari alla base del fenomeno sono state studiate proprietà cinetiche e molecolari delle 3 isoforme NMNAT note da mammifero. Tale studio ha permesso di mettere a punto un saggio di determinazione individuale della loro attività applicabile direttamente a estratti tissutali grezzi. Tale saggio consente di rilevare in maniera diretta le alterazioni a carico delle singole isoforme, già osservate in varie patologie cronico-degenerative riguardanti il sistema nervoso, nella pellagra e in stati patologici cellulari definiti “pellagra-like”, strettamente connessi alla carenza nutrizionale della vitamina PP

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

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 ADPribose pyrophosphatase activity, and discuss the functional connection of such an activity with NAD recycling. An indepth phylogenetic analysis of the COG1058 domain evidenced that in most bacterial species it is fused to PncC, while in a- and some d-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 59-diphosphate and FAD. Multiple sequence alignment analysis, and superposition of the available threedimensional 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 b

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

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

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

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

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