Diguanylate cyclase
Encyclopedia
In enzymology, diguanylate cyclase, also known as diguanylate kinase , is an enzyme
that catalyzes the chemical reaction
:
2 Guanosine triphosphate ↔ 2 diphosphate + cyclic di-3',5'-guanylate
The substrates of diguanylate cyclases (DGCs) are two molecules of guanosine triphosphate
(GTP) and the products are two molecules of diphosphate and one molecule of cyclic di-3’,5’-guanylate (cyclic di-GMP
).
Degradation of cyclic di-GMP to guanosine monophosphate
(GMP) is catalyzed by a phosphodiesterase
(PDE).
sequence motifs “GGDEF
” (Gly-Gly-Asp
-Glu
-Phe
) or “GGEEF” (Gly-Gly-Glu-Glu-Phe), which constitute the domain
of the DGC active site
. These domains are often found coupled to other signaling domains within multidomain proteins. Oftentimes, GGDEF domains with DGC activity are found in the same proteins as c-di-GMP-specific phosphodiesterase (PDE) EAL
(Glu-Ala
-Leu
) domains.
DGC is thought to only be active as a dimer consisting of two subunits
, both with GGDEF domains. The active (or catalytic) site is located at the interface between the two subunits, each binding one molecule of GTP. (See Activation mechanism and Regulation section for more information)
Weak sequence similarity and pronounced secondary structure
similarity between GGDEF domains and the catalytic domains of adenylate cyclase
s (AC) have led to the hypothesis that DGCs and ACs share a similar fold. This was verified with the resolution of the crystal structure
of the DGC PleD from Caulobacter crescentus
in complex with c-di-GMP. As shown in the figure, active PleD, shown as a dimer, is composed of the catalytic DCG domain (labeled DGC) and two CheY-like receiver domains (labeled D1/D2). The DGC domain of each subunit is linked to the two CheY-like domains by a flexible peptide linkage chain. The DCG domain closely resembles the domain of the AC catalytic core which consists of a five-stranded β-sheet surrounded by helices.
As of mid 2011, 11 crystal structures of confirmed or putative DGCs have been solved, with PDB
accession codes , , , , , , , , , , and .
, cyclic-di-GMP, involved in bacterial biofilm
formation and persistence. The GGDEF domain was first identified in the regulatory protein, PleD of the bacterium Caulobacter crescentus
. It was later noted that numerous bacterial genomes encoded multiple proteins with a GGDEF domain. Pseudomonas aeruginosa
PAO1 has 33 proteins with GGDEF domains, Escherichia coli
K-12 has 19, and Vibrio cholerae
O1 has 41. In the cell cycle
of Caulobacter crescentus
, DGC PleD is known to control pole morphogenesis
. In Pseudomonas fluorescens
DGC WspR activity is hypothesized to be partially responsible for the wrinkly spreader (WS) phenotype. In Pseudomonas aeruginosa
, WspR has also been known to control autoaggregation.
Inhibition of DGC activity is thought to be allosteric and non-competitive
. Cyclic di-GMP binds to interface between the DGC and D2 domains stabilizing the open structure and preventing catalysis. Strong product inhibition has been observed with a Ki of 0.5 μM.
Though the exact catalytic mechanism has not been resolved, it is hypothesized that the dimerized structure of PleD facilitates interaction of the two GTP molecules within the DGC active site for cyclization. A proposed mechanism by Chan et al. indicates that the 3'-OH
group of the GTP is deprotonated by a glutamic acid residue (E370) to allow for intermolecular nucleophilic attack of the α-phosphate
. The pentachoordinated transition state created through this nucleophilic attack is possibly stabilized by a Lysine residue (K332).
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that catalyzes the chemical reaction
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
:
2 Guanosine triphosphate ↔ 2 diphosphate + cyclic di-3',5'-guanylate
The substrates of diguanylate cyclases (DGCs) are two molecules of guanosine triphosphate
Guanosine triphosphate
Guanosine-5'-triphosphate is a purine nucleoside triphosphate. It can act as a substrate for the synthesis of RNA during the transcription process...
(GTP) and the products are two molecules of diphosphate and one molecule of cyclic di-3’,5’-guanylate (cyclic di-GMP
Cyclic di-GMP
Cyclic di-GMP is a second messenger used in signal transduction in a wide variety of bacteria. Cyclic di-GMP is not known to be used by eukaryotes or archaea...
).
Degradation of cyclic di-GMP to guanosine monophosphate
Guanosine monophosphate
Guanosine monophosphate, also known as 5'-guanidylic acid or guanylic acid and abbreviated GMP, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside guanosine. GMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase...
(GMP) is catalyzed by a phosphodiesterase
Phosphodiesterase
A phosphodiesterase is any enzyme that breaks a phosphodiester bond. Usually, people speaking of phosphodiesterase are referring to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below...
(PDE).
Structure
Diguanylate cyclases are characterized by the conserved amino acidAmino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
sequence motifs “GGDEF
GGDEF domain
In molecular biology, the GGDEF domain is a protein domain which appears to be ubiquitous in bacteria and is often linked to a regulatory domain, such as a phosphorylation receiver or oxygen sensing domain. Its function is to synthesize cyclic di-GMP, which is used as an intracellular signalling...
” (Gly-Gly-Asp
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
-Glu
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
-Phe
PHE
PHE may refer to:* Population Health and Environment , an approach to development that integrates health or family planning with conservation efforts* Paramount Home Entertainment* BitTorrent protocol encryption...
) or “GGEEF” (Gly-Gly-Glu-Glu-Phe), which constitute the domain
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
of the DGC active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
. These domains are often found coupled to other signaling domains within multidomain proteins. Oftentimes, GGDEF domains with DGC activity are found in the same proteins as c-di-GMP-specific phosphodiesterase (PDE) EAL
EAL domain
In molecular biology, the EAL domain is a conserved protein domain. It is found in diverse bacterial signalling proteins. It is named EAL after its conserved residues. The EAL domain may function as a diguanylate phosphodiesterase. The domain contains many conserved acidic residues that could...
(Glu-Ala
Alanine
Alanine is an α-amino acid with the chemical formula CH3CHCOOH. The L-isomer is one of the 20 amino acids encoded by the genetic code. Its codons are GCU, GCC, GCA, and GCG. It is classified as a nonpolar amino acid...
-Leu
Leucine
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
) domains.
DGC is thought to only be active as a dimer consisting of two subunits
Protein subunit
In structural biology, a protein subunit or subunit protein is a single protein molecule that assembles with other protein molecules to form a protein complex: a multimeric or oligomeric protein. Many naturally occurring proteins and enzymes are multimeric...
, both with GGDEF domains. The active (or catalytic) site is located at the interface between the two subunits, each binding one molecule of GTP. (See Activation mechanism and Regulation section for more information)
Weak sequence similarity and pronounced secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
similarity between GGDEF domains and the catalytic domains of adenylate cyclase
Adenylate cyclase
Adenylate cyclase is part of the G protein signalling cascade, which transmits chemical signals from outside the cell across the membrane to the inside of the cell ....
s (AC) have led to the hypothesis that DGCs and ACs share a similar fold. This was verified with the resolution of the crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
of the DGC PleD from Caulobacter crescentus
Caulobacter crescentus
Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams.Caulobacter is an important model organism for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation. Caulobacter daughter cells have...
in complex with c-di-GMP. As shown in the figure, active PleD, shown as a dimer, is composed of the catalytic DCG domain (labeled DGC) and two CheY-like receiver domains (labeled D1/D2). The DGC domain of each subunit is linked to the two CheY-like domains by a flexible peptide linkage chain. The DCG domain closely resembles the domain of the AC catalytic core which consists of a five-stranded β-sheet surrounded by helices.
As of mid 2011, 11 crystal structures of confirmed or putative DGCs have been solved, with PDB
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....
accession codes , , , , , , , , , , and .
Biological function
Diguanylate cyclase participate in the formation of the ubiquitous second messengerSecond messenger system
Second messengers are molecules that relay signals from receptors on the cell surface to target molecules inside the cell, in the cytoplasm or nucleus. They relay the signals of hormones like epinephrine , growth factors, and others, and cause some kind of change in the activity of the cell...
, cyclic-di-GMP, involved in bacterial biofilm
Biofilm
A biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance...
formation and persistence. The GGDEF domain was first identified in the regulatory protein, PleD of the bacterium Caulobacter crescentus
Caulobacter crescentus
Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams.Caulobacter is an important model organism for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation. Caulobacter daughter cells have...
. It was later noted that numerous bacterial genomes encoded multiple proteins with a GGDEF domain. Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many...
PAO1 has 33 proteins with GGDEF domains, Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
K-12 has 19, and Vibrio cholerae
Vibrio cholerae
Vibrio cholerae is a Gram-negative, comma-shaped bacterium. Some strains of V. cholerae cause the disease cholera. V. cholerae is facultatively anaerobic and has a flagella at one cell pole. V...
O1 has 41. In the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
of Caulobacter crescentus
Caulobacter crescentus
Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams.Caulobacter is an important model organism for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation. Caulobacter daughter cells have...
, DGC PleD is known to control pole morphogenesis
Morphogenesis
Morphogenesis , is the biological process that causes an organism to develop its shape...
. In Pseudomonas fluorescens
Pseudomonas fluorescens
Pseudomonas fluorescens is a common Gram-negative, rod-shaped bacterium. It belongs to the Pseudomonas genus; 16S rRNA analysis has placed P. fluorescens in the P. fluorescens group within the genus, to which it lends its name....
DGC WspR activity is hypothesized to be partially responsible for the wrinkly spreader (WS) phenotype. In Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many...
, WspR has also been known to control autoaggregation.
Role of DGC in C. crescentus cell cycle
During the cell cycle of "C. cresentus", proteins with GGDEF and EAL domains are separated towards the two distinct poles. The active form of diguanylate cyclase PleD localizes to the stalked pole of differentiating C. crescentus cells. It has been suggested that the function of PleD is two-fold. Pled is responsible for turning off flagellum rotations and inhibiting motility before genome replication begins and also for regenerating motility after differentiation has completed.Activation Mechanism and Regulation
The crystal structure of the C. crescentus diguanylate cyclase, PleD, contains three domains; a GGDEF domain with diguanylate cyclase activity and two CheY-like receiver domains (D1/D2). As seen in the figure, the active form of PleD is a dimer which forms by phosphorylation of the first receiver domain (D1). Phosphorylation of the receiver domain increases the dimerization affinity by approximately 10-fold over non-phosphorylated domains.Inhibition of DGC activity is thought to be allosteric and non-competitive
Non-competitive inhibition
Non-competitive inhibition is a type of enzyme inhibition where the inhibitor reduces the activity of the enzyme, by binding not to the active site on the enzyme, but to a different site...
. Cyclic di-GMP binds to interface between the DGC and D2 domains stabilizing the open structure and preventing catalysis. Strong product inhibition has been observed with a Ki of 0.5 μM.
Though the exact catalytic mechanism has not been resolved, it is hypothesized that the dimerized structure of PleD facilitates interaction of the two GTP molecules within the DGC active site for cyclization. A proposed mechanism by Chan et al. indicates that the 3'-OH
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
group of the GTP is deprotonated by a glutamic acid residue (E370) to allow for intermolecular nucleophilic attack of the α-phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
. The pentachoordinated transition state created through this nucleophilic attack is possibly stabilized by a Lysine residue (K332).