Application | Comment | Organism |
---|---|---|
molecular biology | generation of structural models for product-inhibited, elongated dimer | Pseudomonas aeruginosa |
Cloned (Comment) | Organism |
---|---|
from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) | Pseudomonas fluorescens |
from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) | Pseudomonas aeruginosa |
from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) | Pseudomonas putida |
from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3) | Pseudomonas syringae |
Crystallization (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
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 |
L167D | predominantly monomeric, weakly associated dimer, purifies cyclic di-3,5-guanylate-bound, inactive (also upon cyclic di-3,5-guanylate hydrolysis) | Pseudomonas aeruginosa |
L170D | highly active also in cyclic di-3,5-guanylate-bound state, in compact dimer-tetramer equilibrium like nucleotide-free wild-type | Pseudomonas aeruginosa |
R198/242A | double mutant of inhibitory site RxxD, highly active | Pseudomonas aeruginosa |
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 |
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 | Comment | Organism | Structure |
---|---|---|---|
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 | |
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 | |
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 | |
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] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
39170 | - |
theoretical, hexa-His-tagged wild-type | Pseudomonas aeruginosa |
39170 | - |
wild-type, LC-ESI-MS | Pseudomonas aeruginosa |
55000 | - |
mutant L167D, gel-filtration, monomer or weakly associated dimer | Pseudomonas aeruginosa |
73300 | - |
mutant L170D, coupled gel-filtration/multiangle light scattering, tetramer | Pseudomonas aeruginosa |
78600 | - |
mutant R242A, coupled gel-filtration/multiangle light scattering, dimer | Pseudomonas aeruginosa |
80100 | - |
mutant R198/242A, coupled gel-filtration/multiangle light scattering, dimer | Pseudomonas aeruginosa |
81300 | - |
mutant R198A, coupled gel-filtration/multiangle light scattering, dimer | Pseudomonas aeruginosa |
81300 | - |
wild-type (phosphodiesterase-treated) and GGAAF mutant, coupled gel-filtration/multiangle light scattering, dimer | Pseudomonas aeruginosa |
85900 | - |
wild-type, coupled gel-filtration/multiangle light scattering, dimer | Pseudomonas aeruginosa |
161200 | - |
mutant R198/242A, coupled gel-filtration/multiangle light scattering, tetramer | Pseudomonas aeruginosa |
161300 | - |
mutant L170D, coupled gel-filtration/multiangle light scattering, tetramer | Pseudomonas aeruginosa |
161600 | - |
mutant R198A, coupled gel-filtration/multiangle light scattering, tetramer | Pseudomonas aeruginosa |
162100 | - |
mutant R242A, coupled gel-filtration/multiangle light scattering, tetramer | Pseudomonas aeruginosa |
162700 | - |
GGAAF mutant, coupled gel-filtration/multiangle light scattering, tetramer | Pseudomonas aeruginosa |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | - |
PAO1 | - |
Pseudomonas fluorescens | - |
PfO-1 | - |
Pseudomonas fluorescens PfO-1 | - |
PfO-1 | - |
Pseudomonas putida | - |
KT2440 | - |
Pseudomonas putida KT 2240 | - |
KT2440 | - |
Pseudomonas syringae | - |
DC3000 | - |
Pseudomonas syringae DC3000 | - |
DC3000 | - |
Purification (Comment) | Organism |
---|---|
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 |
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 |
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 |
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 Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3,5-guanylate thus reduction of product inhibition | Pseudomonas fluorescens |
additional information | - |
increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3,5-guanylate thus reduction of product inhibition | Pseudomonas aeruginosa |
additional information | - |
increased activity in presence of phosphodiesterases through hydrolysis of cyclic di-3,5-guanylate thus reduction of product inhibition | Pseudomonas putida |
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 | Organism |
---|---|
-80°C, 25 mM Tris-HCl pH 7.5 including 100 mM NaCl and 1 mM dithiothreitol | Pseudomonas aeruginosa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | ? | |
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 | ? | |
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 | ? | |
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 | ? | |
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 | ? | |
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 | ? | |
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 | ? | |
additional information | pppGpG co-purified with reaction products, possible intermediate of diguanylate cyclisation | Pseudomonas aeruginosa | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
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 |
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 |
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 |
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 |
dimer | elongated shape, product-inhibited state, purified wild-type (cyclic di-3,5-guanylate-bound), coupled gel-filtration/multiangle light scattering | Pseudomonas fluorescens |
dimer | elongated shape, product-inhibited state, purified wild-type (cyclic di-3,5-guanylate-bound), coupled gel-filtration/multiangle light scattering | Pseudomonas aeruginosa |
dimer | elongated shape, product-inhibited state, purified wild-type (cyclic di-3,5-guanylate-bound), coupled gel-filtration/multiangle light scattering | Pseudomonas putida |
dimer | elongated shape, product-inhibited state, purified wild-type (cyclic di-3,5-guanylate-bound), coupled gel-filtration/multiangle light scattering | Pseudomonas syringae |
More | compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively | Pseudomonas fluorescens |
More | compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively | Pseudomonas aeruginosa |
More | compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively | Pseudomonas putida |
More | compact and elongated dimeric conformation correspond to catalytically active and inactive state, respectively | Pseudomonas syringae |
More | higher-order oligomerisation depends on interactions of stalk motifs | Pseudomonas aeruginosa |
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 |
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 |
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 |
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 | Comment | Organism |
---|---|---|
diguanylate cyclase | - |
Pseudomonas fluorescens |
diguanylate cyclase | - |
Pseudomonas aeruginosa |
diguanylate cyclase | - |
Pseudomonas putida |
diguanylate cyclase | - |
Pseudomonas syringae |
WspR | locus tag PA3702 | Pseudomonas aeruginosa |
WspR | locus tag Pfl__1058 | Pseudomonas fluorescens |
WspR | locus tag PP__1494 | Pseudomonas putida |
WspR | locus tag PSPTO__1499 | Pseudomonas syringae |