Literature summary extracted from
Fuchs, E.L.; Brutinel, E.D.; Klem, E.R.; Fehr, A.R.; Yahr, T.L.; Wolfgang, M.C.
In vitro and in vivo characterization of the Pseudomonas aeruginosa cAMP phosphodiesterase CpdA required for cAMP homeostasis and virulence factor regulation (2010), J. Bacteriol., 192, 2779-2790.
Cloned(Commentary)
EC Number |
Cloned (Comment) |
Organism |
---|
3.1.4.53 |
expressed in Escherichia coli M15(pREP4) cells |
Pseudomonas aeruginosa |
3.1.4.53 |
gene cpdA, DNA and mino acid sequenc determination and analysis, expression of His-tagged enzyme in Escherichia coli strain M15(pREP4) |
Pseudomonas aeruginosa |
Protein Variants
EC Number |
Protein Variants |
Comment |
Organism |
---|
3.1.4.53 |
D63A |
the mutant shows less than 0.1% of wild type CpdA activity |
Pseudomonas aeruginosa |
3.1.4.53 |
H23A |
the mutant shows less than 0.1% of wild type CpdA activity |
Pseudomonas aeruginosa |
3.1.4.53 |
N93A |
the mutant shows less than 0.1% of wild type CpdA activity |
Pseudomonas aeruginosa |
Inhibitors
EC Number |
Inhibitors |
Comment |
Organism |
Structure |
---|
3.1.4.53 |
alpha-alpha'-dipyridyl |
treatment of CpdA with the Fe2+-specific chelator alpha-alpha'-dipyridyl results in a nearly complete loss of activity |
Pseudomonas aeruginosa |
|
KM Value [mM]
EC Number |
KM Value [mM] |
KM Value Maximum [mM] |
Substrate |
Comment |
Organism |
Structure |
---|
3.1.4.53 |
0.0067 |
- |
3',5'-cAMP |
the addition of FeCl2 does not significantly influence substrate affinity of CdpA increases the rate of the 5'-AMP production, pH and temperature not specified in the publication |
Pseudomonas aeruginosa |
|
Metals/Ions
EC Number |
Metals/Ions |
Comment |
Organism |
Structure |
---|
3.1.4.53 |
Fe2+ |
Fe2+ is required for enzyme activity. The catalytic mechanism for CpdA utilizes a Fe3+-Fe2+ center. Addition of 0.01 mM FeCl2, as a source of Fe2+, results in an 2fold stimulation of CpdA activity |
Pseudomonas aeruginosa |
|
3.1.4.53 |
Fe2+ |
iron and conserved residues are essential for CpdA activity |
Pseudomonas aeruginosa |
|
3.1.4.53 |
additional information |
the addition of Mg2+, Mn2+, Zn2+, and Ca2+ has no effect on CdpA |
Pseudomonas aeruginosa |
|
3.1.4.53 |
additional information |
no effect on enzyme activity by Mg2+, Mn2+, Zn2+, and Ca2+ |
Pseudomonas aeruginosa |
|
Molecular Weight [Da]
EC Number |
Molecular Weight [Da] |
Molecular Weight Maximum [Da] |
Comment |
Organism |
---|
3.1.4.53 |
31000 |
- |
1 * 31000 Da, SDS-PAGE |
Pseudomonas aeruginosa |
Natural Substrates/ Products (Substrates)
EC Number |
Natural Substrates |
Organism |
Comment (Nat. Sub.) |
Natural Products |
Comment (Nat. Pro.) |
Rev. |
Reac. |
---|
3.1.4.53 |
3',5'-cAMP + H2O |
Pseudomonas aeruginosa |
CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro |
5'-AMP |
- |
? |
|
3.1.4.53 |
additional information |
Pseudomonas aeruginosa |
iron and conserved residues are essential for CpdA activity, the catalytic mechanism for Pseudomonas aeruginosa CpdA utilizes a Fe3+-Fe2+ center |
? |
- |
? |
|
Organism
EC Number |
Organism |
UniProt |
Comment |
Textmining |
---|
3.1.4.53 |
Pseudomonas aeruginosa |
D4P095 |
gene cpdA |
- |
3.1.4.53 |
Pseudomonas aeruginosa |
D4P095 |
strain PAK |
- |
Purification (Commentary)
EC Number |
Purification (Comment) |
Organism |
---|
3.1.4.53 |
Ni-NTA column chromatography and Sephacryl S-200 gel filtration |
Pseudomonas aeruginosa |
3.1.4.53 |
recombinant His-tagged enzyme from Escherichia coli strain M15(pREP4) by nickel affinity chromatography |
Pseudomonas aeruginosa |
Substrates and Products (Substrate)
EC Number |
Substrates |
Comment Substrates |
Organism |
Products |
Comment (Products) |
Rev. |
Reac. |
---|
3.1.4.53 |
3',5'-cAMP + H2O |
CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro |
Pseudomonas aeruginosa |
5'-AMP |
- |
? |
|
3.1.4.53 |
adenosine 3',5'-cyclic phosphate + H2O |
- |
Pseudomonas aeruginosa |
adenosine 5'-phosphate |
- |
? |
|
3.1.4.53 |
additional information |
iron and conserved residues are essential for CpdA activity, the catalytic mechanism for Pseudomonas aeruginosa CpdA utilizes a Fe3+-Fe2+ center |
Pseudomonas aeruginosa |
? |
- |
? |
|
Subunits
EC Number |
Subunits |
Comment |
Organism |
---|
3.1.4.53 |
monomer |
1 * 31000 Da, SDS-PAGE |
Pseudomonas aeruginosa |
Synonyms
EC Number |
Synonyms |
Comment |
Organism |
---|
3.1.4.53 |
cAMP phosphodiesterase |
- |
Pseudomonas aeruginosa |
3.1.4.53 |
CpdA |
- |
Pseudomonas aeruginosa |
Temperature Optimum [°C]
EC Number |
Temperature Optimum [°C] |
Temperature Optimum Maximum [°C] |
Comment |
Organism |
---|
3.1.4.53 |
25 |
- |
assay at |
Pseudomonas aeruginosa |
pH Optimum
EC Number |
pH Optimum Minimum |
pH Optimum Maximum |
Comment |
Organism |
---|
3.1.4.53 |
7.6 |
- |
assay at |
Pseudomonas aeruginosa |
Expression
EC Number |
Organism |
Comment |
Expression |
---|
3.1.4.53 |
Pseudomonas aeruginosa |
the cAMP-dependent transcription factor Vfr directly activates expression of cpdA in response to elevated intracellular cAMP |
up |
3.1.4.53 |
Pseudomonas aeruginosa |
cpdA expression is positively regulated by cAMP-Vfr. cAMP-Vfr binds to the cpdA promoter region, suggesting that in vivo, cpdA transcription is directly activated by cAMP-Vfr |
up |
General Information
EC Number |
General Information |
Comment |
Organism |
---|
3.1.4.53 |
malfunction |
deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of Pseudomonas aeruginosa virulence traits |
Pseudomonas aeruginosa |
3.1.4.53 |
malfunction |
deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of Pseudomonas aeruginosa virulence traits. The cpdA mutant has a cAMP-independent small-colony, slow-growth phenotype |
Pseudomonas aeruginosa |
3.1.4.53 |
physiological function |
CpdA is required for cAMP homeostasis and virulence factor regulation, CpdA affects vfr expression and Vfr protein levels and production of virulence factors ExoS, ToxA, and protease IV |
Pseudomonas aeruginosa |
3.1.4.53 |
physiological function |
CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. The cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression. CpdA affects vfr expression and Vfr protein levels. CpdA affects production of virulence factors ExoS, ToxA, and protease IV |
Pseudomonas aeruginosa |