Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase

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.