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Literature summary for 2.7.7.65 extracted from

  • Dahlstrom, K.M.; Giglio, K.M.; Sondermann, H.; OToole, G.A.
    The inhibitory site of a diguanylate cyclase is a necessary element for interaction and signaling with an effector protein (2016), J. Bacteriol., 198, 1595-1603 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.7.7.65

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of the enzyme in Escherichia coli strain BTH101, recombinant expression of enzyme GcbC wild-type and mutants D413K, E360R, and E429R in Pseudomonas aeruginosa strain UCBPP-PA14, the expression is induced with 0.1% arabinose Pseudomonas fluorescens

Crystallization (Commentary)

Crystallization (Comment) Organism
GcbC bound to c-di-GMP, X-ray diffraction structure determination and analysis Pseudomonas fluorescens

Protein Variants

Protein Variants Comment Organism
D413K site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type Pseudomonas fluorescens
E360R site-directed mutagenesis, the mutant shows about 50% increased activity compared to wild-type, but reduced biofilm formation despite elevated levels of c-di-GMP production Pseudomonas fluorescens
E429R site-directed mutagenesis, the mutant shows slightly increased activity compared to wild-type, but reduced biofilm formation despite elevated levels of c-di-GMP production Pseudomonas fluorescens
additional information a mutagenic bacterial two-hybrid (B2H) screen is conducted to identify alleles of GcbC that fail to interact with LapD. Disruption of the I-site of GcbC leads to deregulation of c-di-GMP production Pseudomonas fluorescens
R363E site-directed mutagenesis, the I-site mutant shows a 20fold increase in c-di-GMP production Pseudomonas fluorescens
R366E site-directed mutagenesis, the I-site mutant shows a 16fold increase in c-di-GMP production Pseudomonas fluorescens

Inhibitors

Inhibitors Comment Organism Structure
c-di-GMP feedback inhibition of DGC enzyme GcbC, modelling. In the crystal structure, two molecules of c-di-GMP are found to be bound between the primary and secondary I-sites of this inactive dimer. Three more residues are found to coordinate the formation of the I-site pocket where residue R363 of each monomer of GcbC forms a polar contact with residues E360 and E429 of the other GcbC monomer within the dimer complex of this protein Pseudomonas fluorescens
additional information GcbC has a primary inhibitory site with the previously characterized RXXD motif. The RXXD motif is found at residues R410 to D413 of GcbC. Residue R409 also makes contact with a c-di-GMP molecule. In addition to the primary I-site, GcbC also contains a secondary I-site at position R366 that completes GcbC's regulatory c-di-GMP binding site. The autoinhibitory site (I-site) of a diguanylate cyclase is a necessary element for interaction and signaling with an effector protein Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2 GTP Pseudomonas fluorescens
-
 2 diphosphate + cyclic di-3',5'-guanylate
-
 ?
2 GTP Pseudomonas fluorescens Pfl01
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 2 diphosphate + cyclic di-3',5'-guanylate
-
 ?
additional information Pseudomonas fluorescens diguanylate cyclase GcbC physically interacts with the effector protein LapD in order to promote biofilm formation ?
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additional information Pseudomonas fluorescens Pfl01 diguanylate cyclase GcbC physically interacts with the effector protein LapD in order to promote biofilm formation ?
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-

Organism

Organism UniProt Comment Textmining
Pseudomonas fluorescens Q3K751
-
-
Pseudomonas fluorescens Pfl01 Q3K751
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 GTP
-
 Pseudomonas fluorescens 2 diphosphate + cyclic di-3',5'-guanylate
-
 ?
2 GTP
-
 Pseudomonas fluorescens Pfl01 2 diphosphate + cyclic di-3',5'-guanylate
-
 ?
additional information diguanylate cyclase GcbC physically interacts with the effector protein LapD in order to promote biofilm formation Pseudomonas fluorescens ?
-
-
additional information diguanylate cyclase GcbC physically interacts with the effector protein LapD in order to promote biofilm formation Pseudomonas fluorescens Pfl01 ?
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-

Synonyms

Synonyms Comment Organism
DGC
-
 Pseudomonas fluorescens
DgcP
-
 Pseudomonas fluorescens
gcbC
-
 Pseudomonas fluorescens
Pfl01_4666
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 Pseudomonas fluorescens

General Information

General Information Comment Organism
malfunction mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, it is shown that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. Disruption of the I-site of GcbC leads to deregulation of c-di-GMP production, an increase in the c-di-GMP level and a concomitant increase in c-di-GMP-regulated processes Pseudomonas fluorescens
additional information the autoinhibitory I-site of a diguanylate cyclase is a necessary element for interaction and signaling with an effector protein Pseudomonas fluorescens
physiological function many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity. Signaling specificity in Pseudomonas fluorescens is achieved by physical interaction is the regulation of biofilm formation by the inner membrane proteins GcbC and LapD. GcbC is a DGC that signals to the effector LapD. When bound to c-di-GMP, LapD changes conformation to sequester a periplasmic protease called LapG, thus allowing the large adhesin LapA to accumulate on the cell surface and thereby promoting biofilm formation Pseudomonas fluorescens