The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase.
Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the 'average' enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
Iralis
I
López-Villamizar
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
Alicia
A
Cabezas
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
Rosa María
RM
Pinto
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
José
J
Canales
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
João Meireles
JM
Ribeiro
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
José Carlos
JC
Cameselle
Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain.
María Jesús
MJ
Costas
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D009708:Nucleotidases / Q000235:genetics / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D009712:Nucleotides, Cyclic; D011994:Recombinant Proteins; D009708:Nucleotidases; C040470:3'-nucleotidase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; C000621311:CpdB protein, E coli; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases