title: 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.
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 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.
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Cabezas
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: João Meireles
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Leiria, Leiria, Portugal.
authors.forename: Joaquim Rui
authors.initials: JR
authors.lastname: Rodrigues
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: Iralis
authors.initials: I
authors.lastname: López-Villamizar
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: Ascensión
authors.initials: A
authors.lastname: Fernández
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Canales
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Pinto
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Costas
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
authors.forename: José Carlos
authors.initials: JC
authors.lastname: Cameselle
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D006801:Humans; D008099:Liver / Q000378:metabolism; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D040681:Structural Homology, Protein
chemical_list: D000246:Adenosine Diphosphate Ribose; D008345:Manganese; D017766:Acid Anhydride Hydrolases; D001081:Apyrase; C000603030:ADPRM protein, human