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

• Published in: PloS one 2016-06, Vol.11 (6), p.e0157308-e0157308
• Main Authors: López-Villamizar, Iralis, Cabezas, Alicia, Pinto, Rosa María, Canales, José, Ribeiro, João Meireles, Cameselle, José Carlos, Costas, María Jesús
• Format: Article
• Language: English
• Subjects: 2',3'-Cyclic-Nucleotide Phosphodiesterases - genetics; 2',3'-Cyclic-Nucleotide Phosphodiesterases - metabolism; 3',5'-Cyclic-AMP Phosphodiesterases - genetics; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Amino acid sequence; AMP; Analysis; Annotations; Bacteria; Biofilms; Biology and Life Sciences; Catalysis; Cell cycle; Cellulose; Chemical properties; Cloning; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA fingerprinting; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fingerprinting; Fusion protein; Gel electrophoresis; Genetic aspects; Gram-negative bacteria; Homeostasis; Immune system; Lysates; Mycobacterium tuberculosis; Nucleotidase; Nucleotidases; Nucleotidases - genetics; Nucleotidases - metabolism; Nucleotides, Cyclic - metabolism; Periplasm; Phosphodiesterase; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - metabolism; Physical Sciences; Physiological aspects; Proteins; Recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Research and Analysis Methods; Salmonella; Salmonella enterica; Substrates; Tuberculosis
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0157308

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.