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</p> <B>ABREVIATIONS: </B> ACP – acyl carrier protein, SAM – S-adenosyl-methionine, CoA – coenzyme A </p>
<h2>INTRODUCTION </h2>
<h2>Aim</h2>
<p> For enabling the outside-of-cell activity of the recombinant LuxI enzyme which will serve as the signal transducing element in our system we needed to provide the substrates for it: first S-adenosyl-methionine (SAM) which also serves as a universal donor in methylation reactions and is easy to get from the store and second acylated-acyl carrier protein (in our case hexanoyl-ACP) which is not readily available commercially but IS possible to synthesize by more available strategies. The synthesis and detection of this compound is the topic here. </p>
<p> For enabling the outside-of-cell activity of the recombinant LuxI enzyme which will serve as the signal transducing element in our system we needed to provide the substrates for it: S-adenosyl-methionine (SAM); which also serves as a universal donor in methylation reactions and is easy to get from the store and acylated-acyl carrier protein (in our case hexanoyl-ACP); which is not readily available commercially but is possible to synthesize by more available strategies. The synthesis and detection of this compound is discussed further. </p>
<h2> SYNTHESIS: </h2>
<h2> Synthesis </h2>
<p> There are two major quorum signaling molecules produced by the V. fischeri LuxI enzyme in almost equimolar concentrations: N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone (1) which differ by just a keto group at the third carbon of the acyl chain, however the second compound gives us the great advantage to synthesize it using commercially available products which is why we adopted this approach. </p>
<p> There are two major quorum signaling molecules produced by the <i>V. fischeri</i> LuxI enzyme in almost equimolar concentrations: N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone (1). The two compounds differ only by a keto group at the third carbon of the acyl chain, but hexanoyl homoserine lactone was used since it was advantageous to try synthesizing it using commercially available products.</p>
<p> Normally the in-vivo synthesis of the acyl-ACP substrate for the production of signaling molecules in bacterial quorum sensing is carried out by enzymes like acyl-ACP synthetases which can transfer fatty acid groups onto the functional ACPs (already containing the phosphopantetheine prosthetic group). </p>
<p> Normally the <i> in vivo </i> synthesis of the acyl-ACP substrate for the production of signaling molecules in bacterial quorum sensing is carried out by enzymes like acyl-ACP synthetases which can transfer fatty acid groups onto the functional ACPs (already containing the phosphopantetheine prosthetic group). The two-step purification of the <i> V. fischeri </i> LuxI enzyme and <i> in vitro </i> ACP acylation reactions have been achieved before (2). </p>
<a href="http://2010.igem.org/wiki/images/9/97/ACP1.jpg" rel="lightbox"><img src="http://2010.igem.org/wiki/images/9/97/ACP1.jpg" class="border thumb right"></a></p>
<p> We, however, tried an entirely different synthesis approach by using a different enzyme, namely ACP synthase (4-phosphopantetheinyl-transferase) which is involved in a more general reaction that spans all the domains of life and represents the addition of a prosthetic group to an ACP protein either from the fatty acid or polyketide synthesis pathways in order to make it functional. The prosthetic group in this case is a phosphopantetheine which is taken directly from CoA and added to the apo-protein variant of ACP generating adenosine 3',5'-bisphosphate and holo-ACP (reaction depicted below). </p>
<p> We, however, decided to try an entirely different synthesis approach by using a different enzyme, namely ACP synthase (4-phosphopantetheinyl-transferase) which is involved in a more general reaction that spans all the domains of life and represents the addition of a prosthetic group to an ACP protein either from the fatty acid or polyketide synthesis
pathways in order to make it functional. The prosthetic group in this case is a phosphopantetheine which is taken directly from CoA and added to the apo-protein variant of ACP generating adenosine 3',5'-bisphosphate and holo-ACP (reaction depicted on the right side).
<p> If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety then we would be able to produce hexanoyl-ACP (figure below) ready to be used directly in the reaction with LuxI. </p>
<p> In fact, this is often used in practice for labeling cell surface proteins fused to ACP. (3) </p>
<p> If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety, then we would be able to produce hexanoyl-ACP (figure on the right) ready to be used directly for the <i> in vitro </i> reaction with LuxI. </p>
<p> We tried to produce the reaction using 3 main players: </p>
<p> 1.Hexanoyl CoA trilithium salt hydrate (Sigma-Aldrich) </p>
<p> 2.Acyl carrier protein from E.coli (Sigma-Aldrich) </p>
<p> 3.ACP synthase from E.coli (New England Biolabs) </p>
<p> In fact, the described reaction is a well-established commercial technique used for labeling cell surface proteins fused to ACP with acyl groups of various lengths. (3) </p>
<p> The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix has to be incubated at 37°C for 30 minutes. For terminating the reaction 50 mM EDTA or 10% trichloroacetic acid can be added. </p>
<p> There has been reported mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p>.
</p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p>
</p> As a successful example from literature the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p>
<h2> DETECTION </h2>
<li> Hexanoyl CoA trilithium salt hydrate </li>
<li>Acyl carrier protein HPLC from <i> E.coli </i> – as an alternative for this compound, purified ACP from an <i> E.coli </i> ACPS mutant strain (overexpressing apo-ACP) can be used (8) </li>
<li>ACP synthase from <i> E.coli </i> </li>
<p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo- or holoACP in a SDS-PAGE due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio, thus, facilitating faster migration (fig. 5). (2,7) On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP (fig. 6). </p>
<a href="http://2010.igem.org/wiki/images/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="http://2010.igem.org/wiki/images/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p>
<p> Although this approach isn't quantitative, it would give a hint about the efficiency of the reaction. </p>
<p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p>
<p> A next detection approach is to integrate our system with that of the "yellow" team which would allow to test the success of the fussion protein that they produced. If we test our AHL-sensitive reporter cells with only SAM, our hexanoyl-ACP containing mixture and the recombinant LuxI than the activation of the reporter cells by this cocktail will prove the fact that we have produced both functional acyl-ACP and LuxI enzyme. </p>
</p> The behavior of the ACP synthase depending on the CoA and apo-ACP substrates is illustrated in the figure below. </p>
<a href="http://2010.igem.org/wiki/images/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="http://2010.igem.org/wiki/images/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p>
</p> As a successful example from literature to support our experimental design, the Km value for butyryl production (figure on the right) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p>
<a href="http://2010.igem.org/wiki/images/2/2a/Gel222222.jpg" rel="lightbox"><img src="http://2010.igem.org/wiki/images/2/2a/Gel222222.jpg" class="border thumb right"></a></p>
<p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holo ACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (figure on the right, length of acyl chains is indicated below, "-" stands for holo ACP, 6:0 - for hexanoyl-ACP) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate a band) </p>
<p> An intermediary step for the detection of the acyl-ACP synthesis is the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with SAM, a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to varying amounts of the provided reaction mix containing hexanoyl-ACP can be tracked. Quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis. </p> </p>
<p> A next integrating approach is to test our system with that of the "Fusion protein". If we test our AHL-sensitive reporter cells again with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein (<i>in-vitro</i> activity proved).
<h2> Methods </h2>
<p> The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix is incubated at 37°C for 30 minutes. For terminating the reaction, 50 mM EDTA or 10% trichloroacetic acid is added. </p>
<h2> References </h2>
[[Category:BIOTEC_Dresden/Workflow|ACP]]
An intermediary step for the detection of the acyl-ACP synthesis is the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with SAM, a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to varying amounts of the provided reaction mix containing hexanoyl-ACP can be tracked. Quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis.
A next integrating approach is to test our system with that of the "Fusion protein". If we test our AHL-sensitive reporter cells again with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein (in-vitro activity proved).