Literature summary for 2.7.7.65 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.

Application

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

Cloned(Commentary)

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 (Commentary)

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

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

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

Inhibitors	Comment	Organism
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 [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

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 (Commentary)

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 [micromol/min/mg]

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

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

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