Bifunctional NMN Adenylyltransferase/ADP-Ribose Pyrophosphatase: Structure and Function in Bacterial NAD Metabolism

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Structure (Impact Factor: 5.62). 03/2008; 16(2):196-209. DOI: 10.1016/j.str.2007.11.017
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Bacterial NadM-Nudix is a bifunctional enzyme containing a nicotinamide mononucleotide (NMN) adenylyltransferase and an ADP-ribose (ADPR) pyrophosphatase domain. While most members of this enzyme family, such as that from a model cyanobacterium Synechocystis sp., are involved primarily in nicotinamide adenine dinucleotide (NAD) salvage/recycling pathways, its close homolog in a category-A biodefense pathogen, Francisella tularensis, likely plays a central role in a recently discovered novel pathway of NAD de novo synthesis. The crystal structures of NadM-Nudix from both species, including their complexes with various ligands and catalytic metal ions, revealed detailed configurations of the substrate binding and catalytic sites in both domains. The structure of the N-terminal NadM domain may be exploited for designing new antitularemia therapeutics. The ADPR binding site in the C-terminal Nudix domain is substantially different from that of Escherichia coli ADPR pyrophosphatase, and is more similar to human NUDT9. The latter observation provided new insights into the ligand binding mode of ADPR-gated Ca2+ channel TRPM2.

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Available from: Nadia Raffaelli, Mar 14, 2014
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    • "In turn, the ADPR moiety released from ADP-ribosylated proteins, or deriving from O-acetyl-ADPR and 1,2 cyclic phosphate ADPR, can be further hydrolyzed by ADPR pyrophosphatases (ADPRP) of the Nudix family, yielding AMP and ribose-5-phosphate, which may be reused in NAD biogenesis via conversion to ATP and PRPP, respectively, as already proposed in eukaryotes [13], [14], [15]. In this view, the occurrence in some bacterial species of a Nudix ADPRP domain fused to the enzyme NMN adenylyltransferase (NadM) provides evidence of the strict link between NAD consumption and regeneration (Figure 1) [16], [17]. "
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    ABSTRACT: 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.
    PLoS ONE 06/2013; 8(6):e65595. DOI:10.1371/journal.pone.0065595 · 3.23 Impact Factor
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    • "Apart from these hits, global searches with the C-terminal domain yielded significant matches (E-values ≤ 2.5×10-6) to: nicotinamide mononucleotide (NMN) adenylyl transferase/ribosylnicotinamide kinase from Haemophilus influenzae (pdb 1lw7), ethanolamine-phosphate cytidylyltransferase from H. sapiens (pdb 3elb), nicotinamide-nucleotide adenylyltransferase (pdb 2qjt) from Francisella tularensis and the C-terminal module of bifunctional nicotinamide mononucleotide (NMN) adenylyltransferase/Nudix hydrolase from Synechocystis sp. (SyNadM-Nudix) (pdb 2qjo; [35]). "
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    ABSTRACT: Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity.
    BMC Evolutionary Biology 10/2010; 10(1):311. DOI:10.1186/1471-2148-10-311 · 3.37 Impact Factor
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    • "The details of soNrtR ligand binding site are revealed in its complex structure with ADPR (Fig. 6), which are very similar to that observed in NadM_Nudix proteins but different from other bacterial ADPRases (Huang et al., 2008). The adenine ring of ADPR is sandwiched between the side chains of Phe56 from one subunit and Phe15′ from the second subunit of the dimer. "
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    ABSTRACT: Besides its function as an essential redox cofactor, nicotinamide adenine dinucleotide (NAD) also serves as a consumable substrate for several reactions with broad impact on many cellular processes. NAD homeostasis appears to be tightly controlled, but the mechanism of its regulation is little understood. Here we demonstrate that a previously predicted bacterial transcriptional regulator, NrtR, represses the transcription of NAD biosynthetic genes in vitro. The NAD metabolite ADP-ribose functions as an activator suppressing NrtR repressor activity. The presence of high ADP-ribose levels in the cell is indicative of active NAD turnover in bacteria, which could signal the activation of NAD biosynthetic gene expression via inhibiting the repressor function of NrtR. By comparing the crystal structures of NrtR in complex with DNA and with ADP-ribose, we identified a "Nudix switch" element that likely plays a critical role in the allosteric regulation of DNA binding and repressor function of NrtR.
    Structure 08/2009; 17(7):939-51. DOI:10.1016/j.str.2009.05.012 · 5.62 Impact Factor
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