Information on EC 2.7.7.65 - diguanylate cyclase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.7.65
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RECOMMENDED NAME
GeneOntology No.
diguanylate cyclase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 GTP = 2 diphosphate + cyclic di-3',5'-guanylate
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
nucleotidyl group transfer
-
-
P-O bond cleavage
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SYSTEMATIC NAME
IUBMB Comments
GTP:GTP guanylyltransferase
A GGDEF-domain-containing protein that requires Mg2+ or Mn2+ for activity. The enzyme can be activated by BeF3, a phosphoryl mimic, which results in dimerization [3]. Dimerization is required but is not sufficient for diguanylate-cyclase activity [3]. Cyclic di-3',5'-guanylate is an intracellular signalling molecule that controls motility and adhesion in bacterial cells. It was first identified as having a positive allosteric effect on EC 2.4.1.12, cellulose synthase (UDP-forming) [1].
CAS REGISTRY NUMBER
COMMENTARY hide
146316-82-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain PCC 7120
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-
Manually annotated by BRENDA team
KT2440
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-
Manually annotated by BRENDA team
strain RSP3513
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Manually annotated by BRENDA team
strain RSP3513
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Manually annotated by BRENDA team
bi-functional enzyme with cyclase and phosphodiesterase functions
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 GTP
2 diphosphate + cyclic di-3',5'-guanylate
show the reaction diagram
GTP
cyclic di-3',5'-guanylate + diphosphate
show the reaction diagram
GTP
diphosphate + cyclic di-3',5'-guanylate
show the reaction diagram
GTP + GTP
cyclic di-3',5'-guanylate + diphosphate + diphosphate
show the reaction diagram
GTP + GTP
cyclic-di-3',5'-GMP + diphosphate + diphosphate
show the reaction diagram
pH 7.8
reaction stop with 25 mM EDTA, pH 6, KD (cyclic-di-3’,5’-GMP): 0.3 microM or 0.4 microM (in presence of activator BeF3)
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
GTP
cyclic di-3',5'-guanylate + diphosphate
show the reaction diagram
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diguanylate cyclase activity constitutes the signaling output of the PleD response regulator
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-
?
GTP
diphosphate + cyclic di-3',5'-guanylate
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Li+
-
activity reduced about 20% in 50 mM LiCl
Na+
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50 mM NaCl is used
NaCl
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optimal activity at 300-500 mM NaCl
Zn2+
enzyme is regulated allosterically by zinc. In vitro, zinc reversibly inhibits DgcZ with a subfemtomolar Ki constant. In vivo, bacterial biofilm formation is modulated by externally applied zinc in a DgcZ- and cyclic di-GMP-dependent fashion
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',3'-O-[2,4,6-trinitrocyclohexa-2,5-diene-1,1-diyl]-GTP
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2'-(N-methylanthraniloyl)-GTP
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2'-(N-methylanthraniloyl)-GTP-gamma-S
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3-O-alpha-L-rhamnopyranosyl-(1-2)-beta-D-galactopyranosyl-(1-2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside
4(R)-1-(benzylsulfonyl)-3-phenoxy-2-propanol
4(S)-1-(benzylsulfonyl)-3-phenoxy-2-propanol
cyclic 3',5'-diguanylate
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product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell
cyclic di-3',5'-guanlylate
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strong product inhibition
cyclic di-3',5'-guanylate
cyclic di-GMP
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binding at the inhibitory site mediates dimer formation and inactivation
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cyclic diguanylate
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noncompetitive, product inhibition
cyclic-di-3',5'-GMP
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allosteric feedback, product inhibition, independent of activation status, binding induces change in conformation and protein-solvent interactions
D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside
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isolated from Pisum sativum, specific and highly potent inhibition of enzyme
KCl
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25 mM, slight decrease in activity
N-(((2-phenylethyl)amino)carbonothioyl)benzamide
N-(4-anilinophenyl)benzamide
N-(4-bromophenyl)-3-phenylpropanamide
N-(4-chlorophenyl)-3-phenylpropanamide
N-(4-methoxyphenyl)-3-phenylpropanamide
N-(4-phenoxyphenyl)-2-thiophenecarboxamide
NaCl
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activity decreases with increasing concentrations of NaCl
papulacandin B
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sulfathiazole
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-
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
beryllium fluoride
additional information
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oligomerization-dependent activation of WspR, the stalks are key regulatory elements for the oligomerization, activation, and autoinhibition of WspR
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02 - 0.026
cyclic di-3',5'-guanylate
0.00597 - 0.061
GTP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00081 - 0.016
cyclic di-3',5'-guanylate
0.014 - 59.05
GTP
0.0433 - additional information
cyclic di-3',5'-guanylate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.005
3-O-alpha-L-rhamnopyranosyl-(1-2)-beta-D-galactopyranosyl-(1-2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside
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pH 7.5, 30°C
0.0005 - 0.115
cyclic di-3',5'-guanylate
0.07
papulacandin B
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pH 7.5, 30°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0001
2',3'-O-[2,4,6-trinitrocyclohexa-2,5-diene-1,1-diyl]-GTP
Escherichia coli
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pH 8, 30°C
0.0005
2'-(N-methylanthraniloyl)-GTP
Escherichia coli
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pH 8, 30°C
0.0002
2'-(N-methylanthraniloyl)-GTP-gamma-S
Escherichia coli
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pH 8, 30°C
0.0029 - 0.00817
4(R)-1-(benzylsulfonyl)-3-phenoxy-2-propanol
0.007 - 0.03
GTP
0.0009 - 0.0122
N-(((2-phenylethyl)amino)carbonothioyl)benzamide
0.001 - 0.0178
N-(4-anilinophenyl)benzamide
0.0069 - 0.0175
N-(4-bromophenyl)-3-phenylpropanamide
0.0066 - 0.0135
N-(4-chlorophenyl)-3-phenylpropanamide
0.0098 - 0.03
N-(4-methoxyphenyl)-3-phenylpropanamide
0.008 - 0.0189
N-(4-phenoxyphenyl)-2-thiophenecarboxamide
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000036
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mutant Y26A, in presence of BeF3 (1 mM BeCl2, 10 mM NaF)
0.00004
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mutant Y26A in presence of beryllium trifluoride, pH 7.8
0.0011
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mutant D53N, in presence of BeF3 (1 mM BeCl2, 10 mM NaF); mutant D53N in presence of beryllium trifluoride, pH 7.8
0.00238
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mutant D53N
0.0024
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mutant D53N in absence of beryllium trifluoride, pH 7.8
0.0033
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wild-type in absence of beryllium trifluoride, pH 7.8
0.00332
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wild-type
0.16
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wild-type, in presence of BeF3 (1 mM BeCl2, 10 mM NaF); wild-type in presence of beryllium trifluoride, pH 7.8
151
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pH 10, 60°C, mutant R158A
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 10
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enzymatically active
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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heterocyst differentiation pathway (upstream of HetR), decreased heterocyst frequency and block of heterocyst differentiation within 4 days in mutant strain depleted for all2874 gene and grown in absence of source of combined nitrogen
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
SadC and RoeA are differently localized; SadC and RoeA are differently localized
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251)
Caulobacter crescentus (strain NA1000 / CB15N)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Marinobacter hydrocarbonoclasticus (strain ATCC 700491 / DSM 11845 / VT8)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18000
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DosCH fragment, codon 2-160, determined by SDS-PAGE
21000
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determined by SDS-PAGE
39170
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theoretical, hexa-His-tagged wild-type; wild-type, LC-ESI-MS
50000
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DosC purified from Escherichia coli moderately overexpressing the wild-type dosCP operon, determined by SDS-PAGE
52000
native Anaplasma phagocytophilum PleD, determined by SDS-PAGE and Western Blot analysis; theoretical
55000
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mutant L167D, gel-filtration, monomer or weakly associated dimer
56000
recombinant PleD, determined by SDS-PAGE and Western Blot analysis
73300
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mutant L170D, coupled gel-filtration/multiangle light scattering, tetramer
78000
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x * 78000, calculated
78600
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mutant R242A, coupled gel-filtration/multiangle light scattering, dimer
80100
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mutant R198/242A, coupled gel-filtration/multiangle light scattering, dimer
81300
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mutant R198A, coupled gel-filtration/multiangle light scattering, dimer; wild-type (phosphodiesterase-treated) and GGAAF mutant, coupled gel-filtration/multiangle light scattering, dimer
85900
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wild-type, coupled gel-filtration/multiangle light scattering, dimer
95000
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MBP-DosC, determined by SDS-PAGE
161200
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mutant R198/242A, coupled gel-filtration/multiangle light scattering, tetramer
161300
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mutant L170D, coupled gel-filtration/multiangle light scattering, tetramer
161600
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mutant R198A, coupled gel-filtration/multiangle light scattering, tetramer
162100
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mutant R242A, coupled gel-filtration/multiangle light scattering, tetramer
162700
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GGAAF mutant, coupled gel-filtration/multiangle light scattering, tetramer
170000
determined by Blue-Native PAGE, homotetramer
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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DosCH fragment
homotetramer
monomer
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R158A mutant
multimer
x * 94500, calculated
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
additional information
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pseudo-phosphorylation by beryllium fluoride (BeF3-) modification at residue Asp53 in Rec-domain (D1), tightens dimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
after activation by berillium trifluoride. Activation causes rearrangement of an adaptor domain which in turn promotes dimer formation. The substrate analogue GTPgammaS and two putative cations are bound to the active sites. Identification of a second cyclic diguanylate binding mode that crosslinks the diguanylate cyclase domains within a protein dimer and results in noncompetitive product inhibition; BeF3-/Mg2+-modified PleD (100 microM) in presence of cyclic di-3’,5’-guanylate (0.2 mM) and substrate-analog GTPalphaS (1 mM), hanging-drop vapour-diffusion: equal volumes protein solution (10 mg/ml) and precipitant solution (0.1M HEPES pH 8, 0.73 M Na2SO4), crystals: needle shape, space group: P2(1)2(1)2(1), unit cell parameters: a: 128.9, b: 132.6, c: 88.4, resolved by molecular replacement using PDB: 1W25 as model, tightened dimer interface at the dyad symmetric stem between D1/D2 domains of the two monomers upon rotation of D2 relative to D1, restructured beta4alpha4 loop compared to nonactivated state, GTPalphaS bound to both diguanylate cyclase (GGDEF) domain active sites, 2fold symmetric crosslinking of GGDEF domains of the structural dimer in presence of cyclic di-3’,5’-guanylate, cyclic di-3’,5’-guanylate bound to allosteric (inhibitory) site similarly to nonactivated state
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in complex with product cyclic diguanylate. The guanine base is H-bonded to N335 and D344, whereas the ribosyl and alpha-phosphate moieties extend over the beta2-beta3-hairpin that carries the GGEEF signature motif. In the crystal, cyclic diguanylate molecules are crosslinking active sites of adjacent dimers. In solution, two diguanylate cyclase doamins of a dimer may align in a twofold symmetric way to catalyze synthesis of cyclic diguanylate
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structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF omain in apo and substrate-bound forms. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. Enzyme forms a constitutive dimer and in this form its enzymatic activity is regulated by oxygen binding. The middle domain of DosC connects the sensory and effector modules and is likely to be both essential for and directly involved in the intramolecular signal transduction in DosC
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the zinc ion is bound to the 3His/1Cys motif of the regulatory chemoreceptor zinc-binding domain, which mediates subunit contact within the dimeric enzyme
the crystal structure of G-A1U3W3 (residues 155-312) is determined at 1.83 A resolution using the selenomethionyl SAD method. Co-crystallization with GTP results in enzymatic synthesis of c-di-GMP
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
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to 2.9 A resolution. A peptide loop extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket is observed in the GAF domain
isoform GcbC physically interacting with its target protein at a conserved interface, and this interface can be predictive of diguanylate cyclase-target protein interactions. Physical interaction is necessary for the enzyme to maximally signal its target
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the crystal structure of full-length WspR is determined at 2.8 A resolution, of WspGCN4-RGGDEF at 1.94 A resolution
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structures of an active-like dimeric conformation with both active sites facing each other, of an inactive dimeric conformation, locked by cyclic di-GMP bound at the inhibitory site, and of a single mutant with the R158A mutation at the inhibitory site. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
85.5
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melting temperature, wild-type
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
activity decreases with increasing NaCl concentration and in presence of KCl (25 mM)
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 25 mM Tris-HCl pH 7.5 including 100 mM NaCl and 1 mM dithiothreitol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
application of membrane-free extract on GTP-agarose column
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by Ni-NTA affinity chromatography, furthermore a Superdex 200 and a gel filtration column are used
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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
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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
from bacterial lysate by metal affinity chromatography using HisTrap HP column and FPLC (elution: linear imidazole gradient) and dialysis
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immobilized metal affinity chromatography followed by dialysis and analytical or preparative size exclusion chromatography on Superdex 200 column
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metal affinity chromatography (elution with 200 mM imidazole) followed by size-exclusion chromatography on Superdex 200 HR 26/60 column and analytical size-exclusion chromatography on Superdex 200 HR 10/30 column
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on a Sephadex G25 and a DEAE-Sepharose column, furthermore an amylose-resin matrix is used
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recombinant protein
using a His-Select cartridge
using a TALON metal affinity resin and a Superdex 200 column
using Ni-NTA chromatography
using Ni2+-nitrilotriacetic acid resin and a gel filtration coumn
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
630-bp internal fragment from bacterial DNA in suicide plasmid pRL277 for construction of suicide plasmid pAM4108 and generation of mutant strains by homologous recombination, complete ORF in pAM2770 for generation of pAM4114 for complementation studies, complete ORF in pEZ-30b(+) (pAM4097) for expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3)
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a DNA construct encoding the C-terminus of the TM1788 gene is cloned into the vector pET28b+ for expression in Escherichia col BL21DE3 cells
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addition of a C-terminal His-tag to GcbC results in a loss of function and stability; expressed in Escherichia coli as a His-tagged fusion protein; stably expressed in Escherichia coli as a His-tagged fusion protein
expressed as a His-tagged fusion protein in Escherichia coli
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expressed in Escherichia coli as a His-tagged fusion protein
expression as GFP fusion protein for subcellular targeting, expression with His-tag in Escherichia coli
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expression in a cyanobacterial system
expression in Escherichia coli with His-tag
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expression in Pseudomonas aeruginosa PAO1
expression of MifA or MifB by medium-copy plasmids inhibits motility of Vibrio fischeri. Expression by multicopy plasmids induces further phenotypes correlated with diguanylate cyclase activity such as cellulose biosynthesis and biofilm formation. High-copy expression in Escherichia coli
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for expression with His-tag or as GFP-fusion protein
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from genomic DNA in pET21 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3)
in pBR322 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3)pLysS
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into an Escherichia coli pUC19-based expression vector and into the vector pMAL-c2e
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into the pTOPO vector
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into the vector pET-33b+ for expression in Escherichia coli BL21DE3 cells
into the vector pET3a for expression in Escherichia coli BL21DE3 cells
overexpressed as an amino-terminal MBP fusion in Escherichia coli
overexpressed in Escherichia coli
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overexpression activates cellulose synthesis in Rhizobium leguminosum and in Agrobacterium tumefaciens
overexpression in Escherichia coli as a His-tagged fusion protein is established and allows production of 2.5 mg of pure c-di-GMP per milligram of YdeH with standard biochemical lab equipment
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recombinantly expressed in Escherichia coli. Target protein is produced mainly in the form of inclusion bodies. Both native and mutant enzymes are not inhibited by the target product during conversion of 1 mM GTP into c-di-GMP
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residues 142-579 are expressed as a His-tagged fusion protein in Escherichia coli
the vectors pET21 and pProExHT are used
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D327A
-
inactive due to loss of coordination of Mg2+
D53N
-
less than 1% of wild-type activity; no activation by BeF3, lack of phosphoryl acceptor site
DELTAR359/DELTAD362
mutations in allosteric binding site of cyclic diguanylate, abolish cyclic diguanylate binding, strong decrease in catalytic activity
EE370GG
mutation in active site, complete loss of catalytic activity without effect on allosteric binding of cyclic diguanylate
R148A
increase in allosteric binding of cyclic diguanylate and increase in catalytic activity
R148A/R178A
R148A/R178A/R313A
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triple mutant, KD for binding of cyclic di-3’,5’-guanylate: 4 microM (10fold higher than wild-type), 60fold increased KI for product inhibition by cyclic di-3’,5’-guanylate, residual catalytic activity
R178A
increase in allosteric binding of cyclic diguanylate and increase in catalytic activity
R313A
-
part of inhibitory site
R359A
mutation in allosteric binding site of cyclic diguanylate, strong decrease in cyclic diguanylate binding, strong decrease in catalytic activity
R359V
strong decrease in catalytic activity
R390A
mutation in allosteric binding site of cyclic diguanylate, strong decrease in cyclic diguanylate binding. Catalytic activity comaprable to wild-type
Y26A
-
almost complete loss of activity; dimerisation mutant, nearly inactive, mainly monomeric
D70A
mutated in the response regulator domain, no significant formation of clusters
E253A
mutated in active site, protein forms subcellular clusters in broth-grown cells
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
R198/242A
-
double mutant of inhibitory site RxxD, highly active
V72D
mutated in the response regulator domain, no significant formation of clusters
WspGCN4-RGGDEF
-
fusion protein, coiled-coil segment of GCN4 from Saccharomyces cerevisiae, C-terminal diguanylate cyclase domain with the characteristic GGDEF motif and the active site
WspRD70N
-
mutant, point mutation at the predicted phosphorylation site in the REC domain
WspRGGDEF
-
residues 172-347, C-terminal diguanylate cyclase domain with the characteristic GGDEF motif and the active site
WspRstalk-GGDEF
-
residues 140-347, catalytic activity is modulated by the helical stalk motif, C-terminal diguanylate cyclase domain with the characteristic GGDEF motif and the active site
D287A/E288A
-
mutant has no cyclase activity
E415A
-
mutant has cyclase activity but no phosphodiesterase activity
D177A
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mutation in salt bridge, decrease in melting temperature by 12.3 degrees
E196A
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mutation in salt bridge, decrease in melting temperature by 4.2 degrees
R233A
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mutation in salt bridge, decrease in melting temperature by 8.6 degrees
D177A
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mutation in salt bridge, decrease in melting temperature by 12.3 degrees
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E196A
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mutation in salt bridge, decrease in melting temperature by 4.2 degrees
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R158A
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mutation at the inhibitory site, abolishing product inhibition and unproductive dimerization
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R233A
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mutation in salt bridge, decrease in melting temperature by 8.6 degrees
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D484E
RXXD allosteric inhibition site of the protein is mutated (VC2370(142)-D484E) because it is shown, using HPLC-MS-MS, that c-di-GMP copurifies with native VC2370. Mutation of this RXXD site prevents c-di-GMP copurification. Also, mutation of the site ensures that c-di-GMP produced during the in vitro reaction is not able to inhibit enzyme activity
K759A
mutagenesis of a conserved lysine residue, results in a severe reduction in dihuanylate cyclase activity, kcat value is almost 100fold lower than that of the wild-type protein while the K1 and K2 values do not change very much
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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immobilization of etDGC in sol-gel, a silica matrix that is known to encapsulate biological macromolecules via non-covalent entrapment, is described. The sol-gel matrix preserves the enzymatic activity of the enzyme, and thus, could be a viable method for DGC protein immobilization and c-di-GMP production
medicine
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diguanylate cyclases are interesting targets for new antimicrobial agents with biofilm activity
molecular biology
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generation of structural models for product-inhibited, elongated dimer
synthesis
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the thermophilic enzyme is a valuable tool for c-di-GMP synthesis as well as the preparation of c-di-GMP derivatives
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