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Literature summary for 2.7.7.65 extracted from

  • Wassmann, P.; Chan, C.; Paul, R.; Beck, A.; Heerklotz, H.; Jenal, U.; Schirmer, T.
    Structure of BeF3--modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition (2007), Structure, 15, 915-927.
    View publication on PubMed

Activating Compound

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(Commentary)

Cloned (Comment) Organism
in pBR322 for inducible expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3)pLysS Caulobacter vibrioides

Crystallization (Commentary)

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

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+ enhances dimerisation Caulobacter vibrioides
Mn2+ enhances dimerisation more than Mg2+ Caulobacter vibrioides

Organism

Organism UniProt Comment Textmining
Caulobacter vibrioides
-
isoform PleD
-
Caulobacter vibrioides Q9A5I5
-
-

Posttranslational Modification

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 (Commentary)

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 and Products (Substrate)

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

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

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 [1/s]

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 [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 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