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Literature summary for 2.7.7.65 extracted from

  • Paul, R.; Abel, S.; Wassmann, P.; Beck, A.; Heerklotz, H.; Jenal, U.
    Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization (2007), J. Biol. Chem., 282, 29170-29177.
    View publication on PubMed
Search for more literature for 2.7.7.65

Activating Compound

Activating Compound Comment Organism Structure
beryllium fluoride BeF3, phosphoryl mimic, optimum concentration: 1 mM BeCl2/10 mM NaF (nonspecifcally inhibitory at higher concentrations), causes dimerisation and cyclic di-3’,5’-guanylate synthesis, no influence on cyclic-di-GMP binding affinity and thus allosteric regulation Caulobacter vibrioides

Cloned(Commentary)

Cloned (Comment) Organism
for expression with His-tag or as GFP-fusion protein Caulobacter vibrioides

Protein Variants

Protein Variants Comment Organism
D53N less than 1% of wild-type activity Caulobacter vibrioides
D53N no activation by BeF3, lack of phosphoryl acceptor site Caulobacter vibrioides
E370Q mainly dimeric, inactive mutant of the guanylate cyclase domain GG(D/E)EF (Mg2+ coordination), no activation by BeF3 or protein concentration above 50 microM Caulobacter vibrioides
Y26A almost complete loss of activity Caulobacter vibrioides
Y26A dimerisation mutant, nearly inactive, mainly monomeric Caulobacter vibrioides

General Stability

General Stability Organism
activity decreases with increasing NaCl concentration and in presence of KCl (25 mM) Caulobacter vibrioides

Inhibitors

Inhibitors Comment Organism Structure
cyclic di-3',5'-guanylate product inhibition, allosteric feedback regualtion is not affected by the activation state of enzyme Caulobacter vibrioides
cyclic-di-3',5'-GMP allosteric feedback, product inhibition, independent of activation status, binding induces change in conformation and protein-solvent interactions Caulobacter vibrioides
KCl 25 mM, slight decrease in activity Caulobacter vibrioides
NaCl activity decreases with increasing concentrations of NaCl Caulobacter vibrioides

Localization

Localization Comment Organism GeneOntology No. Textmining
cell pole recruitment to predivisional pole during cell cycle of differentiating cells dependends on dimerisation Caulobacter vibrioides 60187
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
 Caulobacter vibrioides
Mg2+ or Mn2+, strictly required, with Mn2+ resulting in slightly higher activity than Mg2+ Caulobacter vibrioides
Mn2+ or Mg2+, strictly required, with Mn2+ resulting in slightly higher activity than Mg2+ Caulobacter vibrioides
Mn2+ slightly higher activation than Mg2+ Caulobacter vibrioides

Organism

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

Posttranslational Modification

Posttranslational Modification Comment Organism
phosphoprotein enzyme is activated by phosphorylation of residue D53. In vitro, enzyme PleD can be activated by beryllium fluoride, resulting in dimerization and synthesis of cyclic diguanylate Caulobacter vibrioides

Purification (Commentary)

Purification (Comment) Organism
immobilized metal affinity chromatography followed by dialysis and analytical or preparative size exclusion chromatography on Superdex 200 column Caulobacter vibrioides

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 no detectable activity for mutant Y26A Caulobacter vibrioides
0.000036
-
 mutant Y26A, in presence of BeF3 (1 mM BeCl2, 10 mM NaF) Caulobacter vibrioides
0.00004
-
 mutant Y26A in presence of beryllium trifluoride, pH 7.8 Caulobacter vibrioides
0.0011
-
 mutant D53N in presence of beryllium trifluoride, pH 7.8 Caulobacter vibrioides
0.0011
-
 mutant D53N, in presence of BeF3 (1 mM BeCl2, 10 mM NaF) Caulobacter vibrioides
0.00238
-
 mutant D53N Caulobacter vibrioides
0.0024
-
 mutant D53N in absence of beryllium trifluoride, pH 7.8 Caulobacter vibrioides
0.0033
-
 wild-type in absence of beryllium trifluoride, pH 7.8 Caulobacter vibrioides
0.00332
-
 wild-type Caulobacter vibrioides
0.16
-
 wild-type in presence of beryllium trifluoride, pH 7.8 Caulobacter vibrioides
0.16
-
 wild-type, in presence of BeF3 (1 mM BeCl2, 10 mM NaF) Caulobacter vibrioides

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
GTP
-
 Caulobacter vibrioides cyclic di-3',5'-guanylate + diphosphate
-
 ?
GTP + GTP pH 7.8 Caulobacter vibrioides cyclic-di-3',5'-GMP + diphosphate + diphosphate 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) ?

Subunits

Subunits Comment Organism
dimer enzymatically active state, concentration dependent, crosslinking (using disuccinimidyl suberate, DSS) Caulobacter vibrioides
More dimer is the catalytically active state of enzyme Caulobacter vibrioides

Synonyms

Synonyms Comment Organism
diguanylate cyclase
-
 Caulobacter vibrioides
PleD
-
 Caulobacter vibrioides

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 maximum activity between pH 7.5 and 8.5 Caulobacter vibrioides

pH Range

pH Minimum pH Maximum Comment Organism
6.5 10 enzymatically active Caulobacter vibrioides