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

Abstract

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.

Assessing the benefits of horizontal gene transfer by laboratory evolution and genome sequencing

“…Among them is the crp gene, which showed parallel mutations in seven clones from four (one and three ) populations. The c pdA gene and its functional paralogue cpdB [ 76 , 77 ] showed mutations in one and one population (Additional file 25 : Table S9). These genes ( crp, cpdA, cpdB , Additional file 25 : Table S9) are involved in cyclic AMP (cAMP) mediated gene expression regulation.…”

“…Mutations in crp can facilitate survival in low pH [ 79 ], which is highly relevant for our acidic growth condition, and do so probably by derepressing stress response genes. Conversely, cpdA and cpdB encode cAMP phosphodiesterases [ 76 , 77 ] that degrade cAMP. Mutations in the two genes can lead to high cellular cAMP levels [ 80 ].…”

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

“…The hydrolysis of cADPR, NADP + and NAADP were assayed by HPLC measuring respectively the formation of pRibAMP, nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NAMN) as products (see below). The hydrolysis of the other substrates was assayed by colorimetric estimation of the amount of inorganic phosphate 75 liberated from reaction products in the presence of an excess of alkaline phosphatase in the reaction mixture, except that for measuring the activities on ADP and ATP alkaline phosphatase was omitted.…”

“…The reactions were initiated by addition of substrate at the required concentration after a 5-min preincubation of the reaction mixture at 37 °C. Enzyme incubations were terminated either by addition of a phosphate reagent 75 or by injection in a HPLC column (see below). All the assays were run at 37 °C, under conditions of linearity with respect to incubation time and enzyme amount.…”

Molecular Dissection of Escherichia coli CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition, and of the C Domain in Substrate Specificity and Adenosine Inhibition

“…CpdB, the product of the cpdB gene of Escherichia coli , was originally identified as a periplasmic enzyme with phosphohydrolytic activity on 2′,3′-cyclic mononucleotides and 3′-nucleotides [ 1 , 2 , 3 ]. When studied as a recombinant protein, besides showing these activities at a very high catalytic efficiency, CpdB acted also as phosphodiesterase of cyclic dinucleotides, with an efficiency acknowledged to be comparable to other c-di-AMP (3′,5′-cyclic diadenosine monophosphate) or c-di-GMP (3′,5′-cyclic diguanosine monophosphate) phosphodiesterases [ 4 , 5 ]. Cyclic dinucleotide phosphodiesterases include protein (sub)classes characterized by different domain combinations: c-di-GMP-preferring phosphodiesterases bear either an EAL or a HD-GYP domain, and c-di-AMP-preferring phosphodiesterases bear either a HD domain bound to a 7TM receptor or both DHH and DHHA1 domains (reviewed in [ 5 ]).…”