José Canales

Cyclic ADP-ribose (cADPR) is a messenger for Ca²⁺ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe³⁷, Leu¹⁹⁶ and Cys²⁵³ alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys²⁵³ mutation being essential for cADPR preference. Its proximity to the ‘northern’ ribose of cADPR in docking models indicates Cys²⁵³ is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp²⁵⁰, Val²⁵², Cys²⁵³ and Thr²⁷⁹, all near the ‘northern’ ribose. First, the mutant F37A + L196F + C253G, with a smaller residue 253 (Ala > Gly), showed increased cADPR specificity. Then, the mutant F37A + L196F + V252A + C253G, with another residue made smaller (Val > Ala), displayed the desired specificity, with cADPR k_cat/K_M ≈20–200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes.

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 (k_cat/K_m) 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.

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys²⁵³), either within the metallo-dependent phosphatases signature (Gln²⁷, Asn¹¹⁰, His¹¹¹), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe³⁷ and Arg⁴³) and h7h8 (Phe²¹⁰), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe³⁷ was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose K_m and unchanged k_cat of F37A-ADPRibase-Mn, while the K_m values for the other substrates were little affected. Arg⁴³ was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys²⁵³ was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´10⁴ M^-1s^-1 versus 4´10³ M^-1s^-1 of the wild type.

The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn²⁺-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg²⁺ and active with low micromolar Mn²⁺ in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR) in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn²⁺, significant (≈25%) Mg²⁺-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart). The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.

Cyclic 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. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Nucleoside 5′-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25°C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5′-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA _Yi ) of the Y. intermedia ushA gene ( ushA _Yi ) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA _Ec ). Therefore, UshA _Ec was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA _Ec and UshA _Yi behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia , this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k _cat /K _m ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA _Ec conformers and the respective homology models constructed for UshA _Yi , and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis , which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.

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.

An enzyme with FAD-AMP lyase (cyclizing) activity, splitting FAD to AMP and riboflavin 4‘,5‘-phosphate (cFMN), was recently identified [Fraiz, F., et al. (1998) Biochem. J. 330, 881−888]. Now, it has been purified to apparent homogeneity from a rat liver supernatant, by a procedure that includes affinity for ADP-agarose (adsorption required the activating cation Mn²⁺ and desorption required its removal), to a final activity of 2.2 units/mg after a 240-fold purification with a 15% yield. By SDS−PAGE, only one protein band was observed (M_r = 59 000). The correspondence between protein and enzyme activity was demonstrated by renaturation after SDS−PAGE, by gradient ultracentrifugation followed by analytical SDS−PAGE, and by native PAGE with visualization of enzyme activity by fluorescence. A native M_r of 100 000 (ultracentrifugation) or 140 000 (gel filtration) indicated that FAD-AMP lyase could be a dimer. The enzyme required millimolar concentrations of Mn²⁺ or Co²⁺, exhibited different optimum pH values with these cations (pH 8.5 or 7.3, respectively), and was strongly inhibited by ADP or ATP, but not by dADP, dATP, or the reaction products AMP and cFMN. A specificity study was conducted with 35 compounds related to FAD, mostly nucleoside diphosphate-X (NDP-X) derivatives. Besides FAD, the enzyme split 11 of these compounds with the pattern NDP-X → NMP + PX. Structure−activity correlations of substrates, nonsubstrates, and inhibitors, and the comparison of the enzymic reactivities of NDP-X compounds with their susceptibilities to metal-dependent chemical degradation, pinpointed the following specificity pattern. FAD-AMP lyase splits ribonucleoside diphosphate-X compounds in which X is an acyclic or cyclic monosaccharide or derivative bearing an X-OH group that is able to attack internally the proximal phosphorus with the geometry necessary to form a PX product, either a five-atom monocyclic phosphodiester or a cis-bicyclic phosphodiester−pyranose fusion. For instance, NDP-glucose and GDP-α-l-fucose were substrates, but dTDP-glucose, NDP-mannose, and GDP-β-l-fucose were not. Judging from k_cat/K_m ratios, we found the best substrate to be FAD, followed closely by ADP-glucose (k_cat/K_m only 2-fold lower, but not a physiological compound in mammals), whereas other substrates exhibited 50−500-fold lower k_cat/K_m values. However, there was no evidence for specific flavin recognition. Instead, what seems to be recognized is the NDP moiety of NDP-X, with a strong preference for ADP-X. Splitting would then depend on the presence of an adequate X-OH group. The possibility that, besides FAD, there could be in mammals other ADP-X substrates of FAD-AMP lyase is discussed, with emphasis placed on some ADP-ribose derivatives.