Abstract
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.
Highlights
The Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn; EC 3.6.1.53) is one of several enzymes known in mammals to hydrolyze with some degree of specificity the phosphoanhydride linkage of ADP-ribose and other nucleoside diphosphate-X (NDP-X) compounds [1, 2]
Like Aplysia, cyclic ADP-ribose (cADPR) synthesis is catalyzed from NAD+ by an ADP-ribosyl cyclase acting as such [14,15,16], in mammals it is synthesized by a quantitatively minor alternative pathway of the NAD glycohydrolases CD38 and Bst-1/CD157, that mainly hydrolyze NAD+ to ADP-ribose. [17,18,19,20,21,22,23,24]
The turnover of cADPR signaling is exerted by CD38 and Bst-1/CD157, which convert cADPR to ADP-ribose by hydrolysis of the N1-glycosidic linkage [17,18,19, 21, 23]
Summary
The Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn; EC 3.6.1.53) is one of several enzymes known in mammals to hydrolyze with some degree of specificity the phosphoanhydride linkage of ADP-ribose and other nucleoside diphosphate-X (NDP-X) compounds [1, 2]. The ADPRibase-Mn substrates ADP-ribose and cADPR are intracellular signal molecules with known effects on TRPM2 ion channels [5,6,7,8,9] or, in the case of cADPR, on ryanodine receptors [10,11,12] Both are putatively formed, in mammals, from NAD+ by CD38 and Bst-1/ CD157 (reviewed in [13]). It is remarkable that rat ADPRibase-Mn acts as cADPR phosphohydrolase with low but significant efficiency (about 100-fold lower than the activity on ADP-ribose), yielding N1-(5-phosphoribosyl)-AMP as the product. This could hypothetically represent a novel route for cADPR turnover [4]. In the search for pharmacologically-active cADPR analogs [30,31,32,33], the previously overlooked possibility of cADPR phosphohydrolysis is being taken into account [34,35,36]
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