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Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.

ADPRibase-Mn (Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase) was earlier isolated from rat liver supernatants after separation from ADPRibase-I and ADPRibase-II (Mg2+-activated ADP-ribose pyrophosphatases devoid of CDP-alcohol pyrophosphatase activity). The last mentioned are putative Nudix hydrolases, whereas the molecular identity of ADPRibase-Mn is unknown. MALDI (matrix-assisted laser-desorption ionization) MS data from rat ADPRibase-Mn pointed to a hypothetical protein that was cloned and expressed and showed the expected specificity. It is encoded by the RGD1309906 rat gene, which so far has been annotated simply as 'hydrolase'. ADPRibase-Mn is not a Nudix hydrolase, but it shows the sequence and structural features typical of the metallophosphoesterase superfamily. It may constitute a protein family of its own, the members of which appear to be specific to vertebrates, plants and algae. ADP-ribose was successfully docked to a model of rat ADPRibase-Mn, revealing its putative active centre. Microarray data from the GEO (Gene Expression Omnibus) database indicated that the mouse gene 2310004I24Rik, an orthologue of RGD1309906, is preferentially expressed in immune cells. This was confirmed by Northern-blot and activity assay of ADPRibase-Mn in rat tissues. A possible role of ADPRibase-Mn in immune cell signalling is suggested by the second-messenger role of ADP-ribose, which activates TRPM2 (transient receptor potential melastatin channel-2) ion channels as a mediator of oxidative/nitrosative stress, and by the signalling function assigned to many of the microarray profile neighbours of 2310004I24Rik. Furthermore, the influence of ADPRibase-Mn on the CDP-choline or CDP-ethanolamine pathways of phospholipid biosynthesis cannot be discounted.

a b s t r a c tCyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N 1 -glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N 1 -(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.

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