Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn-dependent ADP-ribose/CDP-alcohol diphosphatase.

Cyclic ADP-ribose (cADPR) is a messenger for Ca 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 Phe, Leu and Cys alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys mutation being essential for cADPR preference. Its proximity to the 'northern' ribose of cADPR in docking models indicates Cys is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp, Val, Cys and Thr, 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/K ≈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.