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Literature summary for 3.1.4.52 extracted from

  • Rao, F.; Yang, Y.; Qi, Y.; Liang, Z.X.
    Catalytic Mechanism of c-di-GMP specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa (2008), J. Bacteriol., 190, 3622-3631.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.4.52

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli strain BL21 Pseudomonas aeruginosa
gene PA3947, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pseudomonas aeruginosa

Protein Variants

Protein Variants Comment Organism
D295A inactive Pseudomonas aeruginosa
D295A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
D296A site-directed mutagenesis, the mutant shows a 33.5fold reduced kcat and increased Km compared to the wild-type enzyme Pseudomonas aeruginosa
D296A the mutant shows 33.5fold reduction in turnover number Pseudomonas aeruginosa
D318A site-directed mutagenesis, the mutant shows a reduced kcat and altered Km compared to the wild-type enzyme Pseudomonas aeruginosa
D318A the mutant shows 8.4fold reduction in turnover number Pseudomonas aeruginosa
E175A inactive Pseudomonas aeruginosa
E175A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
E265A inactive Pseudomonas aeruginosa
E265A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
E268A inactive Pseudomonas aeruginosa
E268A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
E268Q site-directed mutagenesis, the mutant shows a reduced kcat and altered Km compared to the wild-type enzyme Pseudomonas aeruginosa
E268Q the mutant shows 446fold reduction in turnover number Pseudomonas aeruginosa
E352A inactive Pseudomonas aeruginosa
E352A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
E352C inactive Pseudomonas aeruginosa
E352C site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
E352D site-directed mutagenesis, the mutant shows a reduced kcat and altered Km compared to the wild-type enzyme Pseudomonas aeruginosa
E352D the mutant shows 30000fold reduction in turnover number Pseudomonas aeruginosa
E352Q site-directed mutagenesis, the mutant shows a reduced kcat and altered Km compared to the wild-type enzyme Pseudomonas aeruginosa
E352Q the mutant shows 61000fold reduction in turnover number Pseudomonas aeruginosa
E355A site-directed mutagenesis, the mutant shows a 1.3fold reduced kcat and increased Km compared to the wild-type enzyme Pseudomonas aeruginosa
E355A the mutant shows 1.3fold reduction in turnover number Pseudomonas aeruginosa
K316A inactive Pseudomonas aeruginosa
K316A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
N233A inactive Pseudomonas aeruginosa
N233A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
Q161A site-directed mutagenesis, the mutant shows increased Km compared to the wild-type enzyme Pseudomonas aeruginosa
Q161A the mutant shows 5.1fold reduction in turnover number Pseudomonas aeruginosa
Q372A site-directed mutagenesis, the mutant shows a reduced kcat and altered Km compared to the wild-type enzyme Pseudomonas aeruginosa
Q372A the mutant shows 8.4fold reduction in turnover number Pseudomonas aeruginosa
R179A site-directed mutagenesis, the mutant shows a 29.1fold reduced kcat and increased Km compared to the wild-type enzyme Pseudomonas aeruginosa
R179A the mutant shows 29.1fold reduction in turnover number Pseudomonas aeruginosa
T267A site-directed mutagenesis, the mutant shows a reduced kcat and altered Km compared to the wild-type enzyme Pseudomonas aeruginosa
T267A the mutant shows 13.4fold reduction in turnover number Pseudomonas aeruginosa

Inhibitors

Inhibitors Comment Organism Structure
Ca2+ Ca2+ ions inhibit the phosphodiesterase activity Pseudomonas aeruginosa
Zn2+ Zn2+ ions inhibit the phosphodiesterase activity Pseudomonas aeruginosa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics of wild-type and mutant RocRs, overview Pseudomonas aeruginosa
0.0003
-
 cyclic-di-GMP mutant enzyme E268Q, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0003
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E268Q Pseudomonas aeruginosa
0.0013
-
 cyclic-di-GMP mutant enzyme Q372A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0013
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant Q372A Pseudomonas aeruginosa
0.0022
-
 cyclic-di-GMP mutant enzyme E352D, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0022
-
 cyclic-di-GMP mutant enzyme T267A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0022
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutants E352D and T267A Pseudomonas aeruginosa
0.0026
-
 cyclic-di-GMP mutant enzyme E355A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0026
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E355A Pseudomonas aeruginosa
0.0032
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant wild-type RocR Pseudomonas aeruginosa
0.0032
-
 cyclic-di-GMP wild type enzyme, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0038
-
 cyclic-di-GMP mutant enzyme E352Q, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0038
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E352Q Pseudomonas aeruginosa
0.0059
-
 cyclic-di-GMP mutant enzyme D318A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0059
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant D318A Pseudomonas aeruginosa
0.0063
-
 cyclic-di-GMP mutant enzyme Q161A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0063
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant Q161A Pseudomonas aeruginosa
0.008
-
 cyclic-di-GMP mutant enzyme R179A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.008
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant R179A Pseudomonas aeruginosa
0.0086
-
 cyclic-di-GMP mutant enzyme D296A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0086
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant D296A Pseudomonas aeruginosa

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ Mn2+ ion can replace Mg2+ ion in catalysis Pseudomonas aeruginosa
Mg2+ required, binding and kinetics, overview Pseudomonas aeruginosa
Mn2+ Mn2+ ion can replace Mg2+ ion in catalysis Pseudomonas aeruginosa

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
42837
-
 4 * 42837, calculated from amino acid sequence, RocR is mainly a tetramer in solution Pseudomonas aeruginosa
176400
-
 calculated from amino acid sequence Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
cyclic di-3',5'-guanylate + H2O Pseudomonas aeruginosa RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg2+ ion 5'-phosphoguanylyl(3'-5')guanosine
-
 ?

Organism

Organism UniProt Comment Textmining
Pseudomonas aeruginosa
-
 gene PA3947
-
Pseudomonas aeruginosa Q9HX69
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni2+-NTA resin column chromatography Pseudomonas aeruginosa
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Pseudomonas aeruginosa

Reaction

Reaction Comment Organism Reaction ID
cyclic di-3',5'-guanylate + H2O = 5'-phosphoguanylyl(3'->5')guanosine hydrolysis of cyclic di-GMP by a general base-catalyzed mechanism with the assistance of a Mg2+ ion, the essential residue E352 functions as a general base catalyst assisting the deprotonation of Mg2+-coordinated water to generate the nucleophilic hydroxide ion, reaction mechanism, residue positioning, and substrate binding, overview Pseudomonas aeruginosa
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cyclic di-3',5'-guanylate + H2O RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg2+ ion Pseudomonas aeruginosa 5'-phosphoguanylyl(3'-5')guanosine
-
 ?
cyclic di-3',5'-guanylate + H2O
-
 Pseudomonas aeruginosa 5'-phosphoguanylyl(3'->5')guanosine
-
 ?
cyclic-di-GMP + H2O
-
 Pseudomonas aeruginosa 5'-pGpG
-
 ?
additional information no hydrolysis of cyclic AMP and cyclic GMP Pseudomonas aeruginosa ?
-
 ?

Subunits

Subunits Comment Organism
More structure modeling and computational docking, overview Pseudomonas aeruginosa
tetramer 4 * 42837, calculated from amino acid sequence, RocR is mainly a tetramer in solution Pseudomonas aeruginosa

Synonyms

Synonyms Comment Organism
c-di-GMP phosphodiesterase
-
 Pseudomonas aeruginosa
cyclic di-GMP-specific phosphodiesterase
-
 Pseudomonas aeruginosa
PDE-A
-
 Pseudomonas aeruginosa
phosphodiesterase A
-
 Pseudomonas aeruginosa
RocR
-
 Pseudomonas aeruginosa
RocR the EAL domain of RocR is catalytically active with cyclic di-GMP-specific phosphodiesterase activity Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
23
-
 assay at Pseudomonas aeruginosa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.000011
-
 cyclic-di-GMP mutant enzyme E352Q, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.000011
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E352Q Pseudomonas aeruginosa
0.000022
-
 cyclic-di-GMP mutant enzyme E352D, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.000022
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E352D Pseudomonas aeruginosa
0.0015
-
 cyclic-di-GMP mutant enzyme E268Q, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.0015
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E268Q Pseudomonas aeruginosa
0.021
-
 cyclic-di-GMP mutant enzyme D296A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.021
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant D296A Pseudomonas aeruginosa
0.023
-
 cyclic-di-GMP mutant enzyme R179A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.023
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant R179A Pseudomonas aeruginosa
0.049
-
 cyclic-di-GMP mutant enzyme T267A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.049
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant T267A Pseudomonas aeruginosa
0.08
-
 cyclic-di-GMP mutant enzyme D318A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.08
-
 cyclic-di-GMP mutant enzyme Q372A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.08
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutants Q372A and D318A Pseudomonas aeruginosa
0.13
-
 cyclic-di-GMP mutant enzyme Q161A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.13
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant Q161A Pseudomonas aeruginosa
0.51
-
 cyclic-di-GMP mutant enzyme E355A, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa
0.51
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant mutant E355A Pseudomonas aeruginosa
0.67
-
 cyclic di-3',5'-guanylate pH 8.0, 23°C, recombinant wild-type RocR Pseudomonas aeruginosa
0.67
-
 cyclic-di-GMP wild type enzyme, in 100 mM Tris buffer (pH 8.0) with 20 mM KCl and 25 mM MgCl2 Pseudomonas aeruginosa

pH Optimum

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

pH Range

pH Minimum pH Maximum Comment Organism
6.2 9.5 pH profiles of recombinant wild-type and mutant E352Q enzymes Pseudomonas aeruginosa