Activating Compound | Comment | Organism | Structure |
---|---|---|---|
beryllium fluoride | BeF3-, pseudo-phosphorylation, enhances dimerisation by lowering KD to less than 10 microM, tightens dimer interface between two D1/D2 domains, modification activates enzyme | Caulobacter vibrioides |
Cloned (Comment) | Organism |
---|---|
in pBR322 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3)pLysS | Caulobacter vibrioides |
Crystallization (Comment) | Organism |
---|---|
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 | Caulobacter vibrioides |
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 | Caulobacter vibrioides |
Protein Variants | Comment | Organism |
---|---|---|
D327A | inactive due to loss of coordination of Mg2+ | Caulobacter vibrioides |
R148A/R178A | double mutant | Caulobacter vibrioides |
R148A/R178A/R313A | 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 | Caulobacter vibrioides |
R313A | part of inhibitory site | Caulobacter vibrioides |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
cyclic di-3',5'-guanylate | noncompetitive product inhibition, binding causes crosslinking of diguanylate cyclase (GGDEF) domains within the dimer, integrity of only one inhibitory site required for inhibition | Caulobacter vibrioides | |
cyclic diguanylate | noncompetitive, product inhibition | Caulobacter vibrioides | |
GTP | - |
Caulobacter vibrioides |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.02 | - |
cyclic di-3',5'-guanylate | double mutant R148A/R178A | Caulobacter vibrioides | |
0.021 | - |
cyclic di-3',5'-guanylate | mutant R313A | Caulobacter vibrioides | |
0.025 | - |
cyclic di-3',5'-guanylate | triple mutant R148A/R178A/R313A | Caulobacter vibrioides | |
0.026 | - |
cyclic di-3',5'-guanylate | wild-type | Caulobacter vibrioides |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | enhances dimerisation | Caulobacter vibrioides | |
Mn2+ | enhances dimerisation more than Mg2+ | Caulobacter vibrioides |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Caulobacter vibrioides | - |
isoform PleD | - |
Caulobacter vibrioides | Q9A5I5 | - |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
additional information | pseudo-phosphorylation by beryllium fluoride (BeF3-) modification at residue Asp53 in Rec-domain (D1), tightens dimer | Caulobacter vibrioides |
Purification (Comment) | Organism |
---|---|
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 | Caulobacter vibrioides |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
GTP + GTP | - |
Caulobacter vibrioides | cyclic di-3',5'-guanylate + diphosphate + diphosphate | analyses by pyrophosphatase-coupled spectrophotometric assay | ? | |
additional information | mode of GTPalphaS/Mg2+-binding suggests two-metal catalytic mechanism analogous to adenylate cyclases | Caulobacter vibrioides | ? | - |
? | |
additional information | on-rates constants of substrate and product range between 100/sec and 300/sec | Caulobacter vibrioides | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | at high protein concentrations and/or presence of divalent metal ions (Mg2+, Mn2+), tightened upon BeF3-modification, exothermic dimerisation, KD: 100 microM, gel filtration chromatography and crystallography | Caulobacter vibrioides |
Synonyms | Comment | Organism |
---|---|---|
diguanylate cyclase | - |
Caulobacter vibrioides |
PleD | response regulator with diguanylate cyclase (GGDEF) domain, Rec domain (D1) and Rec-like adaptor domain (D2) | Caulobacter vibrioides |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
cyclic di-3',5'-guanylate | double mutant R148A/R178A, inhibited, no activity detected | Caulobacter vibrioides | |
additional information | - |
cyclic di-3',5'-guanylate | mutant R313A, inhibited, no activity detected | Caulobacter vibrioides | |
additional information | - |
cyclic di-3',5'-guanylate | wild-type, inhibited, no activity detected | Caulobacter vibrioides | |
0.00081 | - |
cyclic di-3',5'-guanylate | triple mutant R148A/R178A/R313A, inhibited | Caulobacter vibrioides | |
0.0009 | - |
cyclic di-3',5'-guanylate | wild-type | Caulobacter vibrioides | |
0.004 | - |
cyclic di-3',5'-guanylate | mutant R313A | Caulobacter vibrioides | |
0.015 | - |
cyclic di-3',5'-guanylate | double mutant R148A/R178A | Caulobacter vibrioides | |
0.016 | - |
cyclic di-3',5'-guanylate | triple mutant R148A/R178A/R313A | Caulobacter vibrioides |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0005 | - |
cyclic di-3',5'-guanylate | wild-type | Caulobacter vibrioides | |
0.0009 | - |
cyclic di-3',5'-guanylate | mutant R313A | Caulobacter vibrioides | |
0.005 | - |
cyclic di-3',5'-guanylate | double mutant R148A/R178A | Caulobacter vibrioides | |
0.033 | - |
cyclic di-3',5'-guanylate | triple mutant R148A/R178A/R313A | Caulobacter vibrioides |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.007 | - |
wild-type | Caulobacter vibrioides | GTP | |
0.009 | - |
mutant R313A | Caulobacter vibrioides | GTP | |
0.017 | - |
double mutant R148A/R178A | Caulobacter vibrioides | GTP | |
0.03 | - |
triple mutant R148A/R178A/R313A | Caulobacter vibrioides | GTP |