Nadia Raffaelli

Enzymology of NAD+ homeostasis in man.

This review describes the enzymes involved in human pyridine nucleotide metabolism starting with a detailed consideration of their major kinetic, molecular and structural properties. The presentation encompasses all the reactions starting from the de novo pyridine ring formation and leading to nicotinamide adenine dinucleotide (NAD+) synthesis and utilization. The regulation of NAD+ homeostasis with respect to the physiological role played by the enzymes both utilizing NAD+ through the nonredox NAD+-dependent reactions and catalyzing the recycling of the common product, nicotinamide, is discussed. The salient features of other enzymes such as NAD+ pyrophosphatase, nicotinamide mononucleotide 5′-nucleotidase, nicotinamide riboside kinase and nicotinamide riboside phosphorylase, described under ‘miscellaneous’, are likewise presented.

Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase.

A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined. In several cancer cell lines, lower levels of mRNA expression were clearly evident. The gene encoding the human enzyme was mapped to chromosome band 1p32–35. High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium. The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme.

Identification of a novel human nicotinamide mononucleotide adenylyltransferase.

The enzyme nicotinamide mononucleotide adenylyltransferase is an ubiquitous enzyme catalyzing an essential step in NAD (NADP) biosynthetic pathway. In human cells, the nuclear enzyme, which we will now call NMNAT-1, has been the only known enzyme of this type for over 10 years. Here we describe the cloning and expression of a human cDNA encoding a novel 34.4kDa protein, that shares significant homology with the 31.9kDa NMNAT-1. We propose to call this enzyme NMNAT-2. Purified recombinant NMNAT-2 is endowed with NMN and nicotinic acid mononucleotide adenylyltransferase activities, but differs from NMNAT-1 with regard to chromosomal and cellular localization, tissue-specificity of expression, and molecular properties, supporting the idea that the two enzymes might play distinct physiological roles in NAD homeostasis.

Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors

In Gram-negative bacteria, production of bis-(3′,5′)-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.

Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD+ biosynthesis

BACKGROUND Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.

Transcriptional regulation of NAD metabolism in bacteria: NrtR family of Nudix-related regulators

A novel family of transcription factors responsible for regulation of various aspects of NAD synthesis in a broad range of bacteria was identified by comparative genomics approach. Regulators of this family (here termed NrtR for Nudix-related transcriptional regulators), currently annotated as ADP-ribose pyrophosphatases from the Nudix family, are composed of an N-terminal Nudix-like effector domain and a C-terminal DNA-binding HTH-like domain. NrtR regulons were reconstructed in diverse bacterial genomes by identification and comparative analysis of NrtR-binding sites upstream of genes involved in NAD biosynthetic pathways. The candidate NrtR-binding DNA motifs showed significant variability between microbial lineages, although the common consensus sequence could be traced for most of them. Bioinformatics predictions were experimentally validated by gel mobility shift assays for two NrtR family representatives. ADP-ribose, the product of glycohydrolytic cleavage of NAD, was found to suppress the in vitro binding of NrtR proteins to their DNA target sites. In addition to a major role in the direct regulation of NAD homeostasis, some members of NrtR family appear to have been recruited for the regulation of other metabolic pathways, including sugar pentoses utilization and biogenesis of phosphoribosyl pyrophosphate. This work and the accompanying study of NiaR regulon demonstrate significant variability of regulatory strategies for control of NAD metabolic pathway in bacteria.

Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides

Two cytoplasmic forms of pyrimidine nucleotidase (PN‐I and PN‐II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5′‐monophosphates and 3′‐monophosphates respectively. PN‐I and PN‐II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3′‐azido‐3′‐deoxy‐thymidine (AZT), cytosine‐β‐d‐arabinofuranoside (AraC) and 5‐fluoro‐2′‐deoxy‐uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN‐I and PN‐II. PN‐I phosphotransferase activity revealed higher affinity for oxy‐nucleosides with respect to deoxy‐nucleosides, whereas the contrary seems to be true for PN‐II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine‐specific phosphotransferase activity.

Structure and function of nicotinamide mononucleotide adenylyltransferase.

The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily, catalyzes the reaction NMN + ATP = NAD + PPi, representing the final step in the biosynthesis of NAD, a molecule playing a fundamental role as a cofactor in cellular redox reactions. NAD also serves as the substrate for reactions involved in important regulatory roles, such as protein covalent modifications, like ADP-ribosylation reactions, as well as Sir2 histone deacetylase, a recently discovered class of enzymes involved in the regulation of gene silencing. This overview describes the most recent findings on NMNATs from bacteria, archaea, yeast, animal and human sources, with detailed consideration of their major kinetic, molecular and structural features. On this regard, the different characteristics exhibited by the enzyme from the various species are highlighted. The possibility that NMNAT may represent an interesting candidate as a target for the rational design of selective chemotherapeutic agents has been suggested.

Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.

The diguanylate cyclase, Rrp1, regulates critical steps in the enzootic cycle of the Lyme disease spirochetes

Rrp1 is the sole c‐di‐GMP‐producing protein (diguanylate cyclase) of Borrelia burgdorferi. To test the hypothesis that Rrp1 regulates critical processes involved in the transmission of spirochetes between ticks and mammals, an rrp1 deletion mutant (B31‐Δrrp1) and a strain that constitutively produces elevated levels of Rrp1 (B31‐OV) were constructed. The strains were assessed for progression through the enzootic cycle using an Ixodes tick/C3H‐HeJ mouse model and tick immersion feeding methods. B31‐Δrrp1 infected mice as efficiently as wild type but had altered motility, decreased chemotactic responses to N‐acetylglucosamine (NAG) and attenuated ability to disseminate or colonize distal organs. While this strain infected mice, it was not able to survive in ticks. In contrast, B31‐OV displayed normal motility patterns and chemotactic responses but was non‐infectious in mice. Using immersion feeding techniques, we demonstrate that B31‐OV can establish a population in ticks and survive exposure to a natural bloodmeal. The results presented here indicate Rrp1, and by extension, c‐di‐GMP, are not strictly required for murine infection, but are required for the successful establishment of a productive population of B. burgdorferi in ticks. These analyses provide significant new insight into the genetic regulatory mechanisms of the Lyme disease spirochetes.