Identification of the cpdA Gene Encoding Cyclic 3ʹ,5ʹ-Adenosine Monophosphate Phosphodiesterase in Escherichia coli

Abstract

We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3,5-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC 3.1.4.17). The expression of ␤-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene. Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells. Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene. We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP. In addition, we showed that the CpdA protein hydrolyzed cAMP to 5-adenosine monophosphate and that its activity was activated by iron. Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase.cAMP is an important cellular mediator in Escherichia coli. The role of cAMP in mediating glucose effects has been well investigated (1). The cAMP receptor protein (CRP) 1 is a regulatory protein, which binds cAMP and mediates transcriptional regulation at several promoters (2). The CRP-cAMP complex is a positive transcriptional regulator of a number of catabolic operons, including the lac operon in E. coli, and as such, plays a role in catabolite repression, whereby secondary carbon sources are not catabolized in the presence of glucose (3, 4). This complex is involved not only in positive regulation of several catabolic functions but also in regulation of flagellum synthesis (5), toxin production (6), minicell production (7), coupling of DNA replication and cell division (8), and many other functions that are not directly related to catabolism. CRP forms an active conformation only when it binds cAMP. Therefore, the concentration of active CRP-cAMP complex and the biological responses mediated by active CRP-cAMP are influenced by the intracellular concentration of cAMP.Another role of cAMP, which is independent of CRP transcription, has been reported. cAMP interacts directly with the DnaA protein and plays a role in the re-activation of DnaA, which is an essential element for initiation of DNA replication from the chromosome origin, oriC (9). Thus the cellular level of cAMP has some effects on controlling the initiation of DNA replication. Moreover, cAMP has a role in regulation of cell division in E. coli. Filamentation of growing cells is induced by elevated levels of cAMP (10). cAMP may affect cell division only indirectly through unidentified cAMP-dependent functions that are not obligatory (11).In E. coli, the intracellular concentration of cAMP has been thought to be mainly controlled by its own synthesis. Synthesis of cAMP is catalyzed by adenylate cyclase, encoded by the cya gene, and its activity is regulated transcriptionally (12, 13) and post-translationally (14, 15). However, the ex...

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

“…These enzymes are characterized by metal-dependent phosphoesterase activity against a variety of substrates that includes nucleic acids (3)(4)(5), cyclic nucleotides (5)(6)(7)(8), and organophosphates (9). Members of this class of enzymes play crucial roles in diverse cellular processes such as DNA repair, cyclic nucleotide metabolism, and RNA processing.…”

Biochemical Properties of a cAMP Phosphodiesterase in the Cyanobacterium Anabaena sp. strain PCC 7120

“…The Km value for cAMP was 45 mM, which is near the value of E. coli PDE 7) . It is suggested that the PDE functions in cells in which cAMP levels are fairly high, about 40 to 50 mM.…”

“…Figure 4 shows that the PDE activity of the recombinant protein depended upon the presence of 50 mM Fe 2+ and Mn 2+ . The activation of PDE by Fe 2+ has been reported in E. coli 7) . Tertiary structure of CpdA predicted using homology modeling…”

“…The nucleotide sequence of 3',5'-cyclic nucleotide phosphodiesterase of E. coli was used as a query 7) . No homologous gene was found in the Synechocystis genome 9) , but one homologous gene (alr5338) existed in the Anabaena genome 8) .…”

“…Few reports however have been published about the biochemical nature of PDE in prokaryotes. CpdA in Escherichia coli 7) and Icc in Haemophilus influenzae 13) were classified as Class III enzymes, while the periplasmic enzyme CpdP in Vibrio fisheri 5) was assigned to the eukaryotic Class II group.…”