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Literature summary for 3.1.4.53 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.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli M15(pREP4) cells Pseudomonas aeruginosa
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

Protein Variants Comment Organism
D63A the mutant shows less than 0.1% of wild type CpdA activity Pseudomonas aeruginosa
H23A the mutant shows less than 0.1% of wild type CpdA activity Pseudomonas aeruginosa
N93A the mutant shows less than 0.1% of wild type CpdA activity Pseudomonas aeruginosa

Inhibitors

Inhibitors Comment Organism Structure
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]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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

Metals/Ions Comment Organism Structure
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
Fe2+ iron and conserved residues are essential for CpdA activity Pseudomonas aeruginosa
additional information the addition of Mg2+, Mn2+, Zn2+, and Ca2+ has no effect on CdpA Pseudomonas aeruginosa
additional information no effect on enzyme activity by Mg2+, Mn2+, Zn2+, and Ca2+ Pseudomonas aeruginosa

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
31000
-
1 * 31000 Da, SDS-PAGE Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3',5'-cAMP + H2O Pseudomonas aeruginosa CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro 5'-AMP
-
?
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

Organism UniProt Comment Textmining
Pseudomonas aeruginosa D4P095 gene cpdA
-
Pseudomonas aeruginosa D4P095 strain PAK
-

Purification (Commentary)

Purification (Comment) Organism
Ni-NTA column chromatography and Sephacryl S-200 gel filtration Pseudomonas aeruginosa
recombinant His-tagged enzyme from Escherichia coli strain M15(pREP4) by nickel affinity chromatography Pseudomonas aeruginosa

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3',5'-cAMP + H2O CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro Pseudomonas aeruginosa 5'-AMP
-
?
adenosine 3',5'-cyclic phosphate + H2O
-
Pseudomonas aeruginosa adenosine 5'-phosphate
-
?
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

Subunits Comment Organism
monomer 1 * 31000 Da, SDS-PAGE Pseudomonas aeruginosa

Synonyms

Synonyms Comment Organism
cAMP phosphodiesterase
-
Pseudomonas aeruginosa
CpdA
-
Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
assay at Pseudomonas aeruginosa

Expression

Organism Comment Expression
Pseudomonas aeruginosa the cAMP-dependent transcription factor Vfr directly activates expression of cpdA in response to elevated intracellular cAMP up
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

General Information Comment Organism
malfunction deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of Pseudomonas aeruginosa virulence traits Pseudomonas aeruginosa
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
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
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