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Literature summary for 3.1.4.52 extracted from

  • Stelitano, V.; Giardina, G.; Paiardini, A.; Castiglione, N.; Cutruzzola, F.; Rinaldo, S.
    C-di-GMP hydrolysis by Pseudomonas aeruginosa HD-GYP phosphodiesterases: analysis of the reaction mechanism and novel roles for pGpG (2013), PLoS ONE, 8, e74920.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.4.52

Cloned(Commentary)

Cloned (Comment) Organism
commercial synthetic gene PA4108, DNA and amino acid sequence determination and analysis, recombinant overexpression of C- and N-terminally His-tagged enzyme in an endogenous PDE yhjH-lacking Escherichia coli strain and in strain BL21(DE3) Pseudomonas aeruginosa
commercial synthetic gene PA4781, DNA and amino acid sequence determination and analysis, recombinant overexpression of wild-type and mutant C- and N-terminally His-tagged enzymes in an endogenous PDE yhjH-lacking Escherichia coli strain and in strain BL21(DE3), the REC domain of PA4781 cannot be allosterically activated by phosphorylation in the Escherichia coli background Pseudomonas aeruginosa

Protein Variants

Protein Variants Comment Organism
E314A site-directed mutagenesis Pseudomonas aeruginosa
additional information construction of a truncated enzyme version PA4781HD-GYP lacking the REC domain, the recombinant mutant shows highly reduced activity compared to thw wild-type enzyme Pseudomonas aeruginosa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0068
-
 cyclic di-3',5'-guanylate pH 8.0, 30°C, recombinant mutant enzyme E314A Pseudomonas aeruginosa
0.017
-
 5'-phosphoguanylyl(3'->5')guanosine pH 8.0, 30°C, recombinant wild-type enzyme Pseudomonas aeruginosa
0.02
-
 cyclic di-3',5'-guanylate pH 8.0, 30°C, recombinant enzyme Pseudomonas aeruginosa
0.027
-
 5'-phosphoguanylyl(3'->5')guanosine pH 8.0, 30°C, recombinant wild-type enzyme Pseudomonas aeruginosa
0.03
-
 5'-phosphoguanylyl(3'->5')guanosine pH 8.0, 30°C, recombinant enzyme Pseudomonas aeruginosa
0.119
-
 cyclic di-3',5'-guanylate pH 8.0, 30°C, recombinant wild-type enzyme Pseudomonas aeruginosa

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ activates Pseudomonas aeruginosa
Mn2+ activates Pseudomonas aeruginosa

Organism

Organism UniProt Comment Textmining
Pseudomonas aeruginosa Q9HV27 gene PA4781
-
Pseudomonas aeruginosa Q9HWS0 gene PA4108
-

Posttranslational Modification

Posttranslational Modification Comment Organism
phosphoprotein the REC domain of PA4781 cannot be allosterically activated by phosphorylation in the Escherichia coli background, the REC domain at the N-terminus of the protein is responsible for the phosphodiesterase activity of the enzyme. Phosphorylated and non-phosphorylated proteins are both dimeric Pseudomonas aeruginosa

Purification (Commentary)

Purification (Comment) Organism
recombinant C- and N-terminally His-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Pseudomonas aeruginosa
recombinant wild-type and mutant C- and N-terminally His-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Pseudomonas aeruginosa

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5'-phosphoguanylyl(3'->5')guanosine + H2O the isozyme hydrolyses c-di-GMP in a two-step reaction via the linear intermediate 5'-phosphoguanylyl(3'->5')guanosine, it produces GMP in vitro at a low rate, reaction of EC 3.1.4.1 Pseudomonas aeruginosa GMP + guanosine
-
 ?
cyclic di-3',5'-guanylate + H2O the isozyme hydrolyses c-di-GMP in a two-step reaction via the linear intermediate 5'-phosphoguanylyl(3'->5')guanosine, it produces GMP in vitro at a low rate Pseudomonas aeruginosa 5'-phosphoguanylyl(3'->5')guanosine
-
 ?

Subunits

Subunits Comment Organism
dimer phosphorylated and non-phosphorylated proteins are both dimeric Pseudomonas aeruginosa
monomer truncated mutant PA4781HD-GYP protein is mainly a monomer, determined by gel filtration Pseudomonas aeruginosa

Synonyms

Synonyms Comment Organism
HD-GYP PDE
-
 Pseudomonas aeruginosa
PA4108
-
 Pseudomonas aeruginosa
PA4781
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Pseudomonas aeruginosa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.00012
-
 5'-phosphoguanylyl(3'->5')guanosine pH 8.0, 30°C, recombinant enzyme Pseudomonas aeruginosa
0.00015
-
 cyclic di-3',5'-guanylate pH 8.0, 30°C, recombinant enzyme Pseudomonas aeruginosa
0.0002
-
 cyclic di-3',5'-guanylate pH 8.0, 30°C, recombinant wild-type enzyme Pseudomonas aeruginosa
0.0002
-
 5'-phosphoguanylyl(3'->5')guanosine pH 8.0, 30°C, recombinant wild-type enzyme Pseudomonas aeruginosa
0.0006
-
 cyclic di-3',5'-guanylate pH 8.0, 30°C, recombinant mutant enzyme E314A Pseudomonas aeruginosa
0.00077
-
 5'-phosphoguanylyl(3'->5')guanosine pH 8.0, 30°C, recombinant wild-type enzyme Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Pseudomonas aeruginosa

General Information

General Information Comment Organism
malfunction inability of the recombinant Escherichia coli-expressed protein to bind c-di-GMP with high affinity might be due to the nonphosphorylated REC domain which traps the protein in an inactive state, which hampers c-di-GMP binding. Phosphorylation of the REC domain may trigger the conformational change necessary to unmask the active site, thus allowing PA4781 to bind c-di-GMP and enter a catalytic cycle Pseudomonas aeruginosa
additional information the enzyme contains a HD-GYP type catalytic domain Pseudomonas aeruginosa
additional information the enzyme contains a HD-GYP type catalytic domain responsible for Pseudomonas aeruginosa
physiological function cyclic di-3',5'-guanylate is a ubiquitous intracellular second messenger, which is crucial for the physiology and pathogenesis of a variety of bacteria, including those of clinical relevance. C-di-GMP regulates complex prokaryotic processes such as virulence, motility and biofilm formation Pseudomonas aeruginosa