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

Abstract

A gene for cAMP phosphodiesterase, designated cpdA, was identified in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. The predicted amino acid sequence of the gene was similar to the sequences of cAMP phosphodiesterases from Thermosynechococcus elongatus, Escherichia coli and Haemophilus influenzae. The recombinant protein was purified by sequential column chromatography and its biochemical properties were determined. The Anabaena cAMP phosphodiesterase hydrolyzed cAMP and cGMP with similar levels of activity. The K m value for cAMP was 45 mM and the Vmax was 4.9 mmol min -1 mg protein -1 . These values are similar to those of Escherichia coli cAMP phosphodiesterase. The enzyme was activated by divalent cations such as Fe 2+ and Mn 2+ . The tertiary structure of this enzyme was predicted by homology modeling. The deduced structure has two metal-binding sites in the catalytic domain.Key words: cyclic nucleotide phosphodiesterase, cAMP signal transduction, cyanobacteria cAMP is an intracellular second messenger that plays a key role in many physiological processes in both prokaryotes and eukaryotes 3) . cAMP is formed by an adenylate cyclase and hydrolyzed by a cAMP phosphodiesterase. In cyanobacteria, the cellular cAMP level changes in response to several environmental factors such as light-dark, low-high pH, oxic-anoxic and nitrogen repletion-depletion 17,18) . Cells regulate the intracellular concentration of cAMP by balancing its synthesis and degradation. Disruption of an adenylate cyclase gene, cya1, of the unicellular cyanobacterium Synechocystis sp. PCC 6803 caused a decrease in the cellular cAMP level and at the same time immovability of the cells 20) . Overexpression of another adenylate cyclase gene in the filamentous cyanobacterium Anabaena sp. PCC 7120 caused an increase in the cellular cAMP level and fragmentation of the filaments 10) . Thus, not only adenylate cyclase but also phosphodiesterase is important to maintain the cellular concentration of cAMP at an appropriate level.The 3':5'-cyclic nucleotide phosphodiesterase (PDE) [EC 3.1.4.17] catalyzes the hydrolysis of 3':5'-cyclic nucleotides to the corresponding nucleoside 5'-monophosphates. In eukaryotes, PDEs represent a large divergent group of enzymes, and two classes, Class I and Class II, have been recognized 2) . 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.We tried to identify the cyanobacterial PDE gene in the Synechocystis sp. PCC 6803 genome but could not because no sequence characteristic of a hitherto known PDE was detected in this species. In the present study, we searched the

In V itro and In V ivo Characterization of the Pseudomonas aeruginosa Cyclic AMP (cAMP) Phosphodiesterase CpdA, Required for cAMP Homeostasis and Virulence Factor Regulation

“…These results demonstrate that P. aeruginosa CpdA is, in fact, a cAMP phosphodiesterase with an estimated K m of 7.2 Ϯ 1.4 M cAMP and V max of 3.4 Ϯ 0.3 nM 5Ј-AMP produced/min/ng of enzyme. While a proven biological role for 3Ј,5Ј-cyclic GMP (cGMP) in bacteria remains to be determined (26), some class III cyclic nucleotide phosphodiesterases can use both cAMP and cGMP as substrates in vitro (6,15,46). We observed that CpdA of P. aeruginosa also exhibited cGMP phosphodiesterase activity in vitro (see Fig.…”

“…S2), similar to Rv0805 (46) and the CpdA enzyme from the cyanobacterium Anabaena sp. strain PCC 7120 (15). While these enzymes appear to have dual specificity like many class I mammalian cyclic nucleotide phosphodiesterases (6), the relevance of cGMP degradation in P. aeruginosa is unclear, given the lack of measurable cGMP synthesis in vivo.…”

“…Biochemical analyses of cAMP phosphodiesterases from Escherichia coli, Mycobacterium tuberculosis, and Anabaena sp. strain PCC 7120 indicate a similar requirement, suggesting that the structural and functional organization of the catalytic site of class III cyclic nucleotide phosphodiesterases is similar to that of the purple acid phosphatases (15,21,46). E. coli possesses the best-characterized bacterial cAMPdependent signaling system, which primarily controls the metabolic response to environmental carbon availability.…”