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Literature summary extracted from

  • De, N.; Pirruccello, M.; Krasteva, P.V.; Bae, N.; Raghavan, R.V.; Sondermann, H.
    Phosphorylation-independent regulation of the diguanylate cyclase WspR (2008), PLoS Biol., 6, 601-617.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.7.7.65 molecular biology generation of structural models for product-inhibited, elongated dimer Pseudomonas aeruginosa

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.65 from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) Pseudomonas fluorescens
2.7.7.65 from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) Pseudomonas aeruginosa
2.7.7.65 from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) Pseudomonas putida
2.7.7.65 from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) Pseudomonas syringae

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.7.65 in complex with cyclic di-3',5'-guanylate (PDB: 3BRE), solved by molecular replacement using PDB: 1W25 as model, tetramer of two anti-parallel dimers with physically blocked active site, hanging-drop vapour-diffusion: equal volumes protein solution (5-30 mg/ml) and reservoir solution (0.1 M Tris-HCl pH 8, 2.9 M NaCl, 15% xylitol), crystal: space group: C2, unit cell parameter: a: 144.5, b: 72.8, c: 106.1, beta: 110.8, asymmetric unit: two molecules with active site facing each other and four molecules cyclic di-3',5'-guanylate bound to residues Arg242 and Arg198 and two Mg2+ ions, resemblance of GAF domain assembly Pseudomonas aeruginosa

Protein Variants

EC Number Protein Variants Comment Organism
2.7.7.65 GGAAF catalytically dead mutant of active site motif GGEEF, inactive, able to bind cyclic di-3’,5’-guanylate although nucleotide-free when purified, compact dimer-tetramer equilibrium like nucleotide-free wild-type Pseudomonas aeruginosa
2.7.7.65 L167D predominantly monomeric, weakly associated dimer, purifies cyclic di-3’,5’-guanylate-bound, inactive (also upon cyclic di-3’,5’-guanylate hydrolysis) Pseudomonas aeruginosa
2.7.7.65 L170D highly active also in cyclic di-3’,5’-guanylate-bound state, in compact dimer-tetramer equilibrium like nucleotide-free wild-type Pseudomonas aeruginosa
2.7.7.65 R198/242A double mutant of inhibitory site RxxD, highly active Pseudomonas aeruginosa
2.7.7.65 R198A mutant of side chain complementing the inhibitory site R242xxD, highly active, low cyclic di-3’,5’-guanylate affinity, nucleotide-free, in compact dimer-tetramer equilibrium like nucleotide-free wild-type Pseudomonas aeruginosa
2.7.7.65 R242A mutant of inhibitory site RxxD, highly active, low cyclic di-3’,5’-guanylate affinity, nucleotide-free, in compact dimer-tetramer equilibrium like nucleotide-free wild-type Pseudomonas aeruginosa

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.7.65 cyclic di-3',5'-guanylate product inhibition, allosteric, negative feedback, slow intrinsic off-rate of cyclic di-3’,5’-guanylate from RxxD inhibitory-site motif, inhibitory action depends on tetramer formation and subsequent conformational change in dimeric enzyme from global to elongated shape Pseudomonas aeruginosa
2.7.7.65 cyclic di-3',5'-guanylate product inhibition, allosteric, negative feedback, slow intrinsic off-rate of cyclic di-3’,5’-guanylate from RxxD inhibitory-site motif, inhibitory action depends on tetramer formation and subsequent conformational change in dimeric enzyme from global to elongated shape Pseudomonas fluorescens
2.7.7.65 cyclic di-3',5'-guanylate product inhibition, allosteric, negative feedback, slow intrinsic off-rate of cyclic di-3’,5’-guanylate from RxxD inhibitory-site motif, inhibitory action depends on tetramer formation and subsequent conformational change in dimeric enzyme from global to elongated shape Pseudomonas putida
2.7.7.65 cyclic di-3',5'-guanylate product inhibition, allosteric, negative feedback, slow intrinsic off-rate of cyclic di-3’,5’-guanylate from RxxD inhibitory-site motif, inhibitory action depends on tetramer formation and subsequent conformational change in dimeric enzyme from global to elongated shape Pseudomonas syringae

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.7.65 39170
-
 theoretical, hexa-His-tagged wild-type Pseudomonas aeruginosa
2.7.7.65 39170
-
 wild-type, LC-ESI-MS Pseudomonas aeruginosa
2.7.7.65 55000
-
 mutant L167D, gel-filtration, monomer or weakly associated dimer Pseudomonas aeruginosa
2.7.7.65 73300
-
 mutant L170D, coupled gel-filtration/multiangle light scattering, tetramer Pseudomonas aeruginosa
2.7.7.65 78600
-
 mutant R242A, coupled gel-filtration/multiangle light scattering, dimer Pseudomonas aeruginosa
2.7.7.65 80100
-
 mutant R198/242A, coupled gel-filtration/multiangle light scattering, dimer Pseudomonas aeruginosa
2.7.7.65 81300
-
 mutant R198A, coupled gel-filtration/multiangle light scattering, dimer Pseudomonas aeruginosa
2.7.7.65 81300
-
 wild-type (phosphodiesterase-treated) and GGAAF mutant, coupled gel-filtration/multiangle light scattering, dimer Pseudomonas aeruginosa
2.7.7.65 85900
-
 wild-type, coupled gel-filtration/multiangle light scattering, dimer Pseudomonas aeruginosa
2.7.7.65 161200
-
 mutant R198/242A, coupled gel-filtration/multiangle light scattering, tetramer Pseudomonas aeruginosa
2.7.7.65 161300
-
 mutant L170D, coupled gel-filtration/multiangle light scattering, tetramer Pseudomonas aeruginosa
2.7.7.65 161600
-
 mutant R198A, coupled gel-filtration/multiangle light scattering, tetramer Pseudomonas aeruginosa
2.7.7.65 162100
-
 mutant R242A, coupled gel-filtration/multiangle light scattering, tetramer Pseudomonas aeruginosa
2.7.7.65 162700
-
 GGAAF mutant, coupled gel-filtration/multiangle light scattering, tetramer Pseudomonas aeruginosa

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.65 Pseudomonas aeruginosa
-
 PAO1
-
2.7.7.65 Pseudomonas fluorescens
-
 PfO-1
-
2.7.7.65 Pseudomonas fluorescens PfO-1
-
 PfO-1
-
2.7.7.65 Pseudomonas putida
-
 KT2440
-
2.7.7.65 Pseudomonas putida KT 2240
-
 KT2440
-
2.7.7.65 Pseudomonas syringae
-
 DC3000
-
2.7.7.65 Pseudomonas syringae DC3000
-
 DC3000
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.65 from bacterial lysate by metal affinity chromatography and gel-filtration (size-exclusion chromatography), product cyclic di-3’,5’-guanylate co-purified with wild-type and mutants L170D and L167D from bacterial extracts despite dialysis, nucleotide-free wild-type generated by treatment with phosphodiesterase and subsequent metal affinity chromatography and gel-filtration Pseudomonas aeruginosa
2.7.7.65 from bacterial lysate by metal affinity chromatography and gel-filtration, product cyclic di-3’,5’-guanylate co-purified with wild-type from bacterial extracts despite dialysis, nucleotide-free wild-type generated by treatment with phosphodiesterase and subsequent metal affinity chromatography and gel-filtration Pseudomonas fluorescens
2.7.7.65 from bacterial lysate by metal affinity chromatography and gel-filtration, product cyclic di-3’,5’-guanylate co-purified with wild-type from bacterial extracts despite dialysis, nucleotide-free wild-type generated by treatment with phosphodiesterase and subsequent metal affinity chromatography and gel-filtration Pseudomonas putida
2.7.7.65 from bacterial lysate by metal affinity chromatography and gel-filtration, product cyclic di-3’,5’-guanylate co-purified with wild-type from bacterial extracts despite dialysis, nucleotide-free wild-type generated by treatment with phosphodiesterase and subsequent metal affinity chromatography and gel-filtration Pseudomonas syringae

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.7.7.65 additional information
-
 increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3’,5’-guanylate thus reduction of product inhibition Pseudomonas fluorescens
2.7.7.65 additional information
-
 increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3’,5’-guanylate thus reduction of product inhibition Pseudomonas aeruginosa
2.7.7.65 additional information
-
 increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3’,5’-guanylate thus reduction of product inhibition Pseudomonas putida
2.7.7.65 additional information
-
 increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3’,5’-guanylate thus reduction of product inhibition Pseudomonas syringae

Storage Stability

EC Number Storage Stability Organism
2.7.7.65 -80°C, 25 mM Tris-HCl pH 7.5 including 100 mM NaCl and 1 mM dithiothreitol Pseudomonas aeruginosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.65 GTP + GTP 2 mM Mg2+ Pseudomonas aeruginosa cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 GTP + GTP 2 mM Mg2+, 25°C Pseudomonas fluorescens cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 GTP + GTP 2 mM Mg2+, 25°C Pseudomonas putida cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 GTP + GTP 2 mM Mg2+, 25°C Pseudomonas syringae cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 GTP + GTP 2 mM Mg2+, 25°C Pseudomonas syringae DC3000 cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 GTP + GTP 2 mM Mg2+, 25°C Pseudomonas putida KT 2240 cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 GTP + GTP 2 mM Mg2+, 25°C Pseudomonas fluorescens PfO-1 cyclic di-3',5'-guanylate + diphosphate + diphosphate analysis by coupled spectrophotometric assay measuring diphosphate synthesis or by reverse-phase HPLC ?
2.7.7.65 additional information pppGpG co-purified with reaction products, possible intermediate of diguanylate cyclisation Pseudomonas aeruginosa ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.7.65 dimer compact structure, globular shape, active state, phosphodiesterase-treated (hydrolysis of bound cyclic di-3’,5’-guanylate) and nucleotide-free wild-type, coupled gel-filtration/multiangle light scattering Pseudomonas fluorescens
2.7.7.65 dimer compact structure, globular shape, active state, phosphodiesterase-treated (hydrolysis of bound cyclic di-3’,5’-guanylate) and nucleotide-free wild-type, coupled gel-filtration/multiangle light scattering Pseudomonas putida
2.7.7.65 dimer compact structure, globular shape, active state, phosphodiesterase-treated (hydrolysis of bound cyclic di-3’,5’-guanylate) and nucleotide-free wild-type, coupled gel-filtration/multiangle light scattering Pseudomonas syringae
2.7.7.65 dimer compact structure, globular shape, active state, phosphodiesterase-treated (hydrolysis of bound cyclic di-3’,5’-guanylate) and nucleotide-free wild-type, nucleotide-free mutants GGAAF, R242A and R198A and cyclic di-3’,5’-guanylate-bound mutant L170D, coupled gel-filtration/multiangle light scattering Pseudomonas aeruginosa
2.7.7.65 dimer elongated shape, product-inhibited state, purified wild-type (cyclic di-3’,5’-guanylate-bound), coupled gel-filtration/multiangle light scattering Pseudomonas fluorescens
2.7.7.65 dimer elongated shape, product-inhibited state, purified wild-type (cyclic di-3’,5’-guanylate-bound), coupled gel-filtration/multiangle light scattering Pseudomonas aeruginosa
2.7.7.65 dimer elongated shape, product-inhibited state, purified wild-type (cyclic di-3’,5’-guanylate-bound), coupled gel-filtration/multiangle light scattering Pseudomonas putida
2.7.7.65 dimer elongated shape, product-inhibited state, purified wild-type (cyclic di-3’,5’-guanylate-bound), coupled gel-filtration/multiangle light scattering Pseudomonas syringae
2.7.7.65 More compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively Pseudomonas fluorescens
2.7.7.65 More compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively Pseudomonas aeruginosa
2.7.7.65 More compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively Pseudomonas putida
2.7.7.65 More compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively Pseudomonas syringae
2.7.7.65 More higher-order oligomerisation depends on interactions of stalk motifs Pseudomonas aeruginosa
2.7.7.65 tetramer transient intermediate state between active, compact dimer and product-inhibited, elongated dimer, induced by high enzyme concentration, nucleotide-depleted wild-type sample, nucleotide-free mutants GGAAF, R242A and R198A and cyclic di-3’,5’-guanylate-bound mutant L170D, crystallography, coupled gel-filtration/multiangle light scattering Pseudomonas aeruginosa
2.7.7.65 tetramer transient intermediate state between active, compact dimer and product-inhibited, elongated dimer, induced by high enzyme concentration, nucleotide-depleted wild-type, coupled gel-filtration/multiangle light scattering Pseudomonas fluorescens
2.7.7.65 tetramer transient intermediate state between active, compact dimer and product-inhibited, elongated dimer, induced by high enzyme concentration, nucleotide-depleted wild-type, coupled gel-filtration/multiangle light scattering Pseudomonas putida
2.7.7.65 tetramer transient intermediate state between active, compact dimer and product-inhibited, elongated dimer, induced by high enzyme concentration, nucleotide-depleted wild-type, coupled gel-filtration/multiangle light scattering Pseudomonas syringae

Synonyms

EC Number Synonyms Comment Organism
2.7.7.65 diguanylate cyclase
-
 Pseudomonas fluorescens
2.7.7.65 diguanylate cyclase
-
 Pseudomonas aeruginosa
2.7.7.65 diguanylate cyclase
-
 Pseudomonas putida
2.7.7.65 diguanylate cyclase
-
 Pseudomonas syringae
2.7.7.65 WspR locus tag PA3702 Pseudomonas aeruginosa
2.7.7.65 WspR locus tag Pfl__1058 Pseudomonas fluorescens
2.7.7.65 WspR locus tag PP__1494 Pseudomonas putida
2.7.7.65 WspR locus tag PSPTO__1499 Pseudomonas syringae