Sandbox Reserved 1072: Difference between revisions

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OCA, Elizabeth Hughes, Nicole Zimmerman, Geoffrey C. Hoops, David Emch, Isobel Bowles, Jack Trittipo

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 [[Image:C-di-GMP larger font.jpg |200 px|thumb|left|cyclic-dimeric-GMP]]
 [[Image:Poly B-1, 6 GlcNAc.jpg |150 px|left|thumb|poly-β-1,6-N-acetylglucosamine]]
-Diguanylate cyclases are a group of class 2 transferase enzymes that catalyze the production of cyclic dimeric-guanosine monophosphate (c-di-GMP), an important <span class="plainlinks">[https://en.wikipedia.org/wiki/Second_messenger_system second messenger]</span> for <span class="plainlinks">[https://en.wikipedia.org/wiki/Signal_transduction signal transduction]</span>. Signal transduction is the process of sending signals through cells to promote responses, most commonly through phosphorylation or dephosphorylation events. <span class="plainlinks">[https://en.wikipedia.org/wiki/Escherichia_coli ''Escherechia coli'']</span>, a gram-negative bacterium often found in the intestines of mammals, uses DgcZ in the synthesis of its <span class="plainlinks">[https://en.wikipedia.org/wiki/biofilm biofilm]</span>. Enzyme DgcZ from ''E. coli'' acts a catalyst to synthesize cyclic di-GMP from two substrate guanosine triphosphate (GTP) molecules to aid in communication of signals throughout the bacteria. C-di-GMP is a second messenger in the production of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a polysaccharide required for ''E. coli'' biofilm production. This biofilm allows ''E. coli'' to adhere to extracellular surfaces.
+Diguanylate cyclases are a group of class 2 transferase enzymes that catalyze the production of cyclic dimeric-guanosine monophosphate (c-di-GMP), an important <span class="plainlinks">[https://en.wikipedia.org/wiki/Second_messenger_system second messenger]</span> for <span class="plainlinks">[https://en.wikipedia.org/wiki/Signal_transduction signal transduction]</span>. Signal transduction is the process of sending signals through cells to promote responses, most commonly through phosphorylation or dephosphorylation events. <span class="plainlinks">[https://en.wikipedia.org/wiki/Escherichia_coli ''Escherechia coli'']</span>, a gram-negative bacterium often found in the intestines of mammals, uses DgcZ in the synthesis of its <span class="plainlinks">[https://en.wikipedia.org/wiki/biofilm biofilm]</span>. Enzyme DgcZ from ''E. coli'' acts as a catalyst to synthesize cyclic di-GMP from two substrate guanosine triphosphate (GTP) molecules to aid in communication of signals throughout the bacteria. The enzyme has not been crystallized successfully in its active conformation. C-di-GMP is a second messenger in the production of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a polysaccharide required for ''E. coli'' biofilm production. This biofilm allows ''E. coli'' to adhere to extracellular surfaces.
 == Structural Overview ==
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 [[Image:Electrostatic map front view CZB.png|250 px|right|thumb|Electrostatic potential map of the CZB domain of Diguanylate Cyclase. Regions of relatively negative charge are in red and regions of relatively positive charge are in blue. Electrically neutral regions are in white]]
 === Zinc Binding Site ===
-Most cells possess efficient Zinc uptake systems, as Zinc is a reactive Lewis Acid. Zinc binds incredibly tightly to this enzyme at subfemtomolar concentrations. The Zinc co-purified with the protein.Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domain <sup>[1]</sup>. The inhibition prevents regulation of GGDEF domain function, the location of the active site. The CZB domain is folded into four anti-parallel α-helices as a 2-fold symmetric homodimer, with the N-terminus on the helix 𝝰4. The allosteric binding site includes a <scene name='69/694239/Zinc_binding_domain/4'>3His/1Cys</scene> motif that uses amino acids H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, spanning three of the four alpha helices of the CZB domain and coordinating the Zinc residue in a tetrahedral fashion. For clarification, the entirety of 𝝰helix 2 on one monomer of CZB is not successfully crystallized after the Cys52 reside and is not the N-terminal residue.
+Most cells possess efficient Zinc uptake systems, as Zinc is a reactive Lewis Acid. Zinc binds incredibly tightly to this enzyme at subfemtomolar concentrations, attributing to why the enzyme has not been crystallized without Zinc present. The Zinc co-purified with the protein.Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domain <sup>[1]</sup>. The inhibition prevents regulation of GGDEF domain function, the location of the active site. The CZB domain is folded into four anti-parallel α-helices as a 2-fold symmetric homodimer, with the N-terminus on the helix 𝝰4. The allosteric binding site includes a <scene name='69/694239/Zinc_binding_domain/4'>3His/1Cys</scene> motif that uses amino acids H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, spanning three of the four alpha helices of the CZB domain and coordinating the Zinc residue in a tetrahedral fashion. For clarification, the entirety of 𝝰helix 2 on one monomer of CZB is not successfully crystallized after the Cys52 reside and is not the N-terminal residue.
 Zahringer et al. mutated Cys52 to Ala through <span class="plainlinks">[https://en.wikipedia.org/wiki/Site-directed_mutagenesis site-directed mutagenesis]</span>, resulting in a lack of coordination on α2. The cysteine residue is not essential for Zinc binding, as Zinc still coordinates to the three His residues with the Cys52Ala mutation, but α2 is free to move and expose the Zinc binding pocket. This exposure was found to lower the protein's affinity for zinc, as the mutation of cysteine to alanine increased the activity of the DgcZ. Using EDTA, Zinc can be removed from the CZB domain. The zinc has higher affinity for EDTA than CZB when EDTA concentration is higher than the concentration of DgcZ.  When not coordinated to zinc, the CZB domain adopts a conformation that straightens the <scene name='69/694239/Czbd_with_helices_labeled/2'>𝝰1 helix</scene>, shifting <scene name='69/694239/Hydrophobicity_int_residues/3'>hydrophobic residues</scene> on the α-helices into the center and the GGEEF domain into its productive conformation, increasing activity of DgcZ. Activity increases without Zinc due to activation of poly-GlcNAc production and biofilm formation, and maximal cyclic di-GMP production.