Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase
- Authors
- Cabezas, A., Ribeiro, J.M., Rodrigues, J.R., López-Villamizar, I., Fernández, A., Canales, J., Pinto, R.M., Costas, M.J., Cameselle, J.C.
- ID
- ZDB-PUB-150219-2
- Date
- 2015
- Source
- PLoS One 10: e0118680 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Acid Anhydride Hydrolases/chemistry*
- Acid Anhydride Hydrolases/genetics*
- Acid Anhydride Hydrolases/metabolism
- Adenosine Diphosphate Ribose/metabolism*
- Animals
- Apyrase/chemistry*
- Apyrase/genetics*
- Apyrase/metabolism
- Catalytic Domain
- Humans
- Liver/metabolism
- Manganese/metabolism*
- Models, Molecular
- Molecular Docking Simulation
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Rats
- Structural Homology, Protein
- PubMed
- 25692488 Full text @ PLoS One
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 Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km 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.