Team:Leicester/Modeling

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       <li> <a href="/Team:Leicester/Project">Project</a></li>
       <li> <a href="/Team:Leicester/Project">Project</a></li>
       <li> <a href="/Team:Leicester/Chemistry">Chemistry</a></li>
       <li> <a href="/Team:Leicester/Chemistry">Chemistry</a></li>
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       <li> <a href="/Team:Leicester/Parts">Parts Submitted to the Registry</a></li>
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       <li> <a href="/Team:Leicester/Parts">Parts Submitted to Registry</a></li>
       <li> <a href="/Team:Leicester/Modeling">Modeling</a></li>
       <li> <a href="/Team:Leicester/Modeling">Modeling</a></li>
       <li> <a href="/Team:Leicester/Notebook">Notebook</a>
       <li> <a href="/Team:Leicester/Notebook">Notebook</a>
<ul>
<ul>
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        <li> <a href="/Team:Leicester/April2012">April</a></li>
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        <li> <a href="/Team:Leicester/May2012">May</a></li>
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        <li> <a href="/Team:Leicester/June2012">June</a></li>
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         <li> <a href="/Team:Leicester/July2012">July</a></li>
         <li> <a href="/Team:Leicester/July2012">July</a></li>
         <li> <a href="/Team:Leicester/August2012">August</a></li>
         <li> <a href="/Team:Leicester/August2012">August</a></li>
         <li> <a href="/Team:Leicester/September2012">September</a></li>
         <li> <a href="/Team:Leicester/September2012">September</a></li>
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         <li> <a href="/Team:Leicester/October2012">October</a></li>
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         </ul></li>
         </ul></li>
       <li> <a href="/Team:Leicester/Safety">Safety</a></li>
       <li> <a href="/Team:Leicester/Safety">Safety</a></li>
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<li> <a href="/Team:Leicester/HumanPractices">Human Practices</a></li>
       <li> <a href="/Team:Leicester/Attributions">Attributions</a></li>
       <li> <a href="/Team:Leicester/Attributions">Attributions</a></li>
       <li> <a href="/Team:Leicester/Bibliography">Bibliography</a></li>
       <li> <a href="/Team:Leicester/Bibliography">Bibliography</a></li>
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     <h1>Modeling</h1>
     <h1>Modeling</h1>
     <h2>What are we modeling?</h2>
     <h2>What are we modeling?</h2>
-
     <p>The aim of our project is to genetically modify a bacteria that can break down the polymer chains in expanded polystyrene (EPS). The result is a solution of monomer chains that can be extracted and used in the synthetic production of other useful chemicals, for instance Lactic Acid (C<sub>3</sub>H<sub>6</sub>O<sub>3</sub>). The computational model is going to be used to aid calculations predicting how much substrate (polymer chains) will be needed, how much product will be required to sustain the bacteria colony and how much product will be left over for other uses.</p>
+
     <p>The aim of our project is to genetically modify a bacteria that can break down the polymer chains in expanded polystyrene (EPS). The result is a solution of monomer units that can be extracted and used in the synthesis of other useful chemicals, for instance Lactic Acid (C<sub>3</sub>H<sub>6</sub>O<sub>3</sub>). The computational model is going to be used to aid in calculations predicting how much substrate (polymer chains) will be needed, how much product will be required to sustain the bacterial colony and how much product will be left over for other uses.</p>
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/b/b3/ModelingGraphic1.gif" /> </div>
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/b/b3/ModelingGraphic1.gif" /> </div>
      
      
     <h2>Why are we modeling?</h2>
     <h2>Why are we modeling?</h2>
-
     <p>By creating a computational model of a biological system, theoretical and experimental biologists are able to predict the outcomes and behaviour of a system for a set of input parameters. The results of the simulation can provide guidance to the success of the experiment for the tested input parameters before undertaking physically in the lab. The model built in our project will help determine the next steps in the project, for example to attempt to change the number of enzymes produced by the bacteria or modify how the enzyme behaves. The major advantages of developing and using a model in the project are that it can save time exploring an idea that had a high probability of failure and simultaneously save resources.</p>
+
     <p>By creating a computational model of a biological system, theoretical and experimental biologists are able to predict the outcomes and behaviour of a system for a set of input parameters. The results of the simulation can provide guidance on the likely success (or otherwise) of the experiment,for the tested input parameters, before undertaking it physically in the lab. The model built in our project will help determine the next steps in the project, for example to attempt to change the amount of enzyme produced by the bacteria, or to modify how the enzyme behaves. The major advantages of developing and using a model in the project are that it can save time exploring ideas that might have a high probability of failure, while simultaneously saving resources.</p>
<h2>Enzyme Kenetics</h2>
<h2>Enzyme Kenetics</h2>
-
     <p>Enzyme Kinetics is the processes by which chemical reactions are catalysed by enzymes. When we say that we aim to "genetically modify a bacteria that can degrade expanded polystyrene" what we really mean is that we aim to "create a chemical reaction catalysed by enzymes that can break down polystyrene polymer chains". The polymer chains in expanded polystyrene can be broken into monomers by industrial processes, however this involves the use of nasty chemicals. </p>
+
     <p>Enzyme Kinetics is the processes by which chemical reactions are catalysed by enzymes. When we say that we aim to "genetically modify a bacteria that can degrade expanded polystyrene" what we really mean is that we aim to "create a chemical reaction catalysed by enzymes that can break down polystyrene polymer chains". The polymer chains in expanded polystyrene can be broken into monomers by industrial processes, however this involves the use of toxic chemicals. </p>
-
     <p>The method adopted by bacteria to modify their environment, in order to obtain useful products, is the use of enzymes. Enzymes are biological molecules that have the ability to act as a catalyst in chemical reactions, in other words increase the rate of reaction. Enzymes have an active site that fits the substrate, like a "lock and key", once connected the enzyme performs the reaction. The <em>Psuedmonas</em> bacteria in our project secrete enzymes that break the polystyrene polymer in order to extract the carbon. </p>
+
     <p>The method adopted by bacteria to modify their environment to obtain useful products by breakdown of , is the use of enzymes. Enzymes are biological molecules that have the ability to act as a catalyst in chemical reactions. In other words they increase the rate of reaction. Enzymes have an active site that fits the substrate, like a "lock and key"and once connected the enzyme performs the reaction. The <em>Psuedmonas</em> bacteria in our project secrete enzymes that break the polystyrene polymer in order to extract the carbon. </p>
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/8/88/ModelingGraphic2.gif" width="471" height="163"/></div>
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/8/88/ModelingGraphic2.gif" width="471" height="163"/></div>
-
     <p>Above is a diagram illustrating an example of a substrate can be manipulated by the enzyme. The substrate collides with the active site of the enzyme to form a complex, the products are later released by the enzyme.</p>
+
     <p>Above is a diagram illustrating an example of how a substrate can be manipulated by the enzyme. The substrate collides with the active site of the enzyme to form a complex, the products are later released by the enzyme.</p>
-
<p>The formation of substrate to product via the use of enzymes can be described in the following expression: </p>
+
<p>The transformation of substrate to product via the use of enzymes can be described in the following expression: </p>
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/9/95/ModelingGraphic3.gif"/></div>
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/9/95/ModelingGraphic3.gif"/></div>
     <p>Where:<br />
     <p>Where:<br />
Line 58: Line 57:
     <strong>k<sub>-1</sub></strong> is the reverse rate constant for enzyme-substrate formation (the enzyme "drops" the substrate)<br />
     <strong>k<sub>-1</sub></strong> is the reverse rate constant for enzyme-substrate formation (the enzyme "drops" the substrate)<br />
     <strong>k<sub>2</sub></strong> is the forward rate constant for production formation from the enzyme-substrate complex and is assumed irreversible</p>
     <strong>k<sub>2</sub></strong> is the forward rate constant for production formation from the enzyme-substrate complex and is assumed irreversible</p>
 +
 +
<p>The substrate concentration uses the units of molarity. A single monomer chain of polystyrene, known as styrene, has a molecular weight of 104.1 g/mol. In order to get one molarity solution, we would need 104.1 grams in 1Litre of solvent. The average chain molecular weight is approximately 9.7685 x 10<sup>6</sup> g/mol, that's around 93, 838 styrene monomers per average chain. The active sites of the enzymes secreted by the bacteria can only fit the free ends of the polystyrene chain, so out of a 93, 838 long chain of styrene monomers, only 0.002% of the chain is available to the enzyme.</p>
 +
 +
<p>Also, we considered the method the enzyme will use to find the end of the chain. It could either collide with the polymer chain suspended in solution, and at part of the chain hold on and shuffle along to the end - similar to restriction enzymes. The other method, with a chain suspended in solution, specifies that the enzyme can only start catalysing the reaction when it collides with a free end only; a much more lengthly, although possibly more realistic, senerio.</p>
 +
 +
<h2>Michaelis-Menton equation</h2>
 +
    <p>The Michaelis-Menton equation is a model for single-substrate reaction is used as a base for our model. </p>
 +
    <div align="center"><img src="https://static.igem.org/mediawiki/2012/9/9a/MMequation.gif" width="218" height="87" /></div>
      
      
-
<p><a href="/wiki/index.php?title=Team:Leicester/Modeling&amp;action=edit">[edit]</a></p>
+
    <p>Where:<br />
 +
    <strong>V<sub>0</sub></strong> is the initial veliocity of the reaction<br />
 +
    <strong>V<sub>max</sub></strong> is the rate of reaction when substrate is saturated<br />
 +
    <strong>S</strong> is the substrate concentration<br />
 +
    <strong>K<sub>m</sub></strong> is the substrate concentration when the reaction rate is half the maximum and can be calculated using: k<sub>-1</sub> + k<sub>2</sub>/k<sub>1</sub> <br />
 +
    </p>
 +
<h2>Resulting model</h2>
 +
<p>This model simulates enzymes that are released by the bacteria into the solution:</p>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/73/ModelingGraph.jpg" width="800px" /></div>
 +
<div align="center"><p style="font-size:10px"> This model simulates a single bacteria in solution with the polystyrene substrate. Substrate concentration is 1M, where one monomer of Polystyrene is 104.1 g/mol. Also in this model, Tyro is the name of the enzyme digesting Polystyrene</p></div>.
 +
 
 +
<p>In the model the polystyrene degrading enzyme is produced in the cell, and then transported by a specific transporter out of the cell and into the flask. Here it can work on the excess substrate polystyrene, catalysing its conversion to styrene.
 +
<p>This model is not an exact representation of the <em>Psuedomonas</em> behaviour. This is largely due to the fact the values for the M-M equation were gathered from a paper regarding Tyrosinase, but since there was no available data for <em>Psuedomonas</em> using Polystyrene we used this as a model. Further, it has been noted that <em>Pseudomonas</em> do not use free enzymes, but instead rely on the end of a polystyrene chain colliding with the active site on the bacteria [INSERT CITATION?]. </p>
 +
<p>However what this model can tell us, is the pattern of behaviour expected if <em>Psuedomonas</em> was modified to use free enzymes. This can be compared to results gained by observing the <em>Psuedomonas</em> at work, but sadly this data is still currently unavailable to us. </p>
 +
 
 +
</tr>
 +
<tr>
 +
<td><h1>Enzyme modification</h1></td>
 +
</tr>
 +
<tr>
 +
<td><p/>Programs, such as <a href="http://pymol.org/" target="_blank">Pymol</a> , allow one to manipulate the tertiary/quaternary structure of enzymes and other proteins, that have already had their structure determined by X-ray diffraction and/or NMR after they have been successfully crystallised. Whereas programs such as <a href="http://rasmol.org/OpenRasMol.html" target="_blank">RASWIN</a> only allow you to display these structures (in many different ways), Pymol takes this one stage further by giving one the power to manipulate individual residues to change the rotamer (direction/position the atoms in the residue) or even the residue itself (a kind of <i> In silico </i> mutagenesis). This means you can modify enzymes to such an extent that you may be able to fit any substrate you want into a particular enzyme's active site. However, in reality, the modified protein may adopt a completely different tertiary/quaternary structure which may cause it not to actually work as expected. </p>
 +
<p> Polystyrene doesn't have its own degradation pathway due to the short time polystyrene has actually been in any great quantity in the world. Also, polystyrene is extremely unreactive, due to stable phenyl side chains present. Styrene, the polystyrene monomer has a known degradation pathway since being a much smaller molecule, it can diffuse into bacterial cells. The environment of the cell allows much more flexibility with proteins, as the highly reducing internal environment allows unstable complexes to persist (such as iron-sulphur complexes). </p>
 +
<p> Firstly, we identified possible pathways that could be modified to accept polystyrene as a substrate. There were 2 potential pathways we could use: the styrene degradation pathway, and the toluene degrading pathway. Both have several different degradation pathways depending on the bacterial species/strain, but we ended up choosing the aerobic toluene degradation pathway from <i>Pseudomonas putida</i>, as the active sites of the pathway could remove/break the unreactive phenyl groups from the polystyrene chain.   
 +
<p><div align="center"><img src="https://static.igem.org/mediawiki/2012/1/17/Toluene_degading_pathway.jpg"  width="750" height="303.4" alt="Toluene degradation pathway in <i> P. putida</i>" /></div></p>
 +
<p style="font-size:10px;"> The toluene degradation pathway. TDO is the enzyme we modified in silico using Pymol, and is Toluene 2,3-dioxygenase, which is made up of an alpha and beta subunit (TodC1 and TodC2) in a hexamer, with TodA and TodB acting as electron shuttles for the reaction from NADH to the enzyme. TodD is <i>cis</i>-1,2-dihydrobenzene-1,2-diol dehydrogenase which forms 3-methyl catechol. The other enzymes weren't focused on very much, though it was later found that there was no TodD crystal structure available online to modify. (George <i>et. al</i>, 2011)</p>
 +
<p> Toluene 2,3-dioxygenase is the first enzyme of the pathway, and crystal structures are available online on the <a href="http://www.rcsb.org/pdb/home/home.do">RCSB Protein Data bank</a>. It was found that there was a gap directly into the active site, that if widened, could potentially fit polystyrene:
 +
<p><div align="center"><img src="https://0olkfq.bay.livefilestore.com/y1mTqn3QGen6O_MK6uTEKNlxTKpUMhczgGmPGiVTLRYRKKY5J6n7f6z_u2Ld0kIlK6bARqfSt3Kjwc3cwl1t8ovS2XLM051JM5X/3EN1%20pic.png?psid=1"  width="480" height="360" /></div></p>
 +
<p style="font-size:10px;">Toluene 2,3-dioxygenase is made up of alpha and beta subunits. The sticks show each non-hydrogen atom, and the dots show the area each atom takes up. The pink residues indicate the active site residues, and the orange molecule is toluene. As can be seen, there is only a small gap when the enzyme has toluene bound that links the active site to the external environment (from 3EN1). A interactive version of 3EN1 is available <a href="http://www.rcsb.org/pdb/explore/jmol.do?structureId=3EN1&bionumber=1">Here</a></p>
 +
<p>However, there are a few residues that cause this gap to be so small, which we modified to amino acids with smaller sidechains, but that were still hydrophobic residues to widen the gap, potentially allowing polystyrene into the active site. The residues we modified were Met220→Ala220 (which when the Toluene 2,3-dioxygenase was blast searched, came up as a natural variation in some dioxygenases), Val421→Ala421, Tyr422→Leu422 and Tyr266→Val266, which created a bigger gap, which when the polystyrene structure was added to the active site and was sculpted, barely changed the active site structure as can be seen below: </p>
 +
<div align="center"><table cellpadding="0" width="400" cellspacing="0"><tr>
 +
<td><img src="https://static.igem.org/mediawiki/2012/9/96/Modified_1.png" width="480" height="360" border="0"></td>
 +
<td><img src="https://static.igem.org/mediawiki/2012/e/ec/Modified_2.png" width="480" height="360" border="0"></td></tr></table></div>
 +
<div align="center"><p style="font-size:10px;"> Modified Toluene 2,3-dioxygenase enzyme with a similar view to the image above. Again, the pink residues are the active site residues. The white residues are those that have been modified. (left) The polystyrene substrate has been omitted for clarity, but note the gap is much bigger now (modified from 3EN1). (right) The polystyrene is present here: note the very tight fit into the gap </p></div>
 +
 +
<p><div align="center"><img src="https://static.igem.org/mediawiki/2012/3/32/Modified_3.png"  width="480" height="360" /></div></p>
 +
<div align="center"><p style="font-size:10px;">A side view of the modified enzyme. Note that the terminal phenyl group is in the active site <p></div>
 +
<p><div align="center"><img src="https://static.igem.org/mediawiki/2012/8/88/Modified_4.png"  width="480" height="360" /></div></p>
 +
<div align="center"><p style="font-size:10px;"> This view is just of the toluene 2,3-dioxygenase active site (blue), overlapped with the modified version (pink). Notice how similar the active site shape is, which means that potentially, the active site could still catalyse the dioxygenase reaction.</p></div>
 +
<p> Whether or not the relatively large change in the positioning/angle of the polystyrene aromatic ring in the active site would hinder the reaction or not needs to be studied. Because there are also 2 proteins involved in transporting electrons to the enzyme, as few of the modified residues, if any are on the surface, so shouldn't affect binding. A future project could actually get this synthesised and assayed to see whether the modifications could allow polystyrene to bind in the active site, and cause hydroxylation of the side chain. </p>
 +
<h2>References</h2>
 +
<p>Guazzaroni, M., et al, A Novel Synthesis of Bioactive Catechols by Layer-by-Layer Immobilized Tyrosinase in an Organic Solvent Medium, <em>ChemCatChem</em>, 4, 89-99, 2012.</p>
 +
 
 +
 
   </div>
   </div>
    
    

Latest revision as of 01:11, 27 September 2012

iGEM Leicester Test Page 2012

Modeling

What are we modeling?

The aim of our project is to genetically modify a bacteria that can break down the polymer chains in expanded polystyrene (EPS). The result is a solution of monomer units that can be extracted and used in the synthesis of other useful chemicals, for instance Lactic Acid (C3H6O3). The computational model is going to be used to aid in calculations predicting how much substrate (polymer chains) will be needed, how much product will be required to sustain the bacterial colony and how much product will be left over for other uses.

Why are we modeling?

By creating a computational model of a biological system, theoretical and experimental biologists are able to predict the outcomes and behaviour of a system for a set of input parameters. The results of the simulation can provide guidance on the likely success (or otherwise) of the experiment,for the tested input parameters, before undertaking it physically in the lab. The model built in our project will help determine the next steps in the project, for example to attempt to change the amount of enzyme produced by the bacteria, or to modify how the enzyme behaves. The major advantages of developing and using a model in the project are that it can save time exploring ideas that might have a high probability of failure, while simultaneously saving resources.

Enzyme Kenetics

Enzyme Kinetics is the processes by which chemical reactions are catalysed by enzymes. When we say that we aim to "genetically modify a bacteria that can degrade expanded polystyrene" what we really mean is that we aim to "create a chemical reaction catalysed by enzymes that can break down polystyrene polymer chains". The polymer chains in expanded polystyrene can be broken into monomers by industrial processes, however this involves the use of toxic chemicals.

The method adopted by bacteria to modify their environment to obtain useful products by breakdown of , is the use of enzymes. Enzymes are biological molecules that have the ability to act as a catalyst in chemical reactions. In other words they increase the rate of reaction. Enzymes have an active site that fits the substrate, like a "lock and key"and once connected the enzyme performs the reaction. The Psuedmonas bacteria in our project secrete enzymes that break the polystyrene polymer in order to extract the carbon.

Above is a diagram illustrating an example of how a substrate can be manipulated by the enzyme. The substrate collides with the active site of the enzyme to form a complex, the products are later released by the enzyme.

The transformation of substrate to product via the use of enzymes can be described in the following expression:

Where:
E is the number of enzymes
S is the substrate concentration
ES is the enzyme-substrate complex
P is the product
k1 is the forward rate constant for enzyme-substrate formation
k-1 is the reverse rate constant for enzyme-substrate formation (the enzyme "drops" the substrate)
k2 is the forward rate constant for production formation from the enzyme-substrate complex and is assumed irreversible

The substrate concentration uses the units of molarity. A single monomer chain of polystyrene, known as styrene, has a molecular weight of 104.1 g/mol. In order to get one molarity solution, we would need 104.1 grams in 1Litre of solvent. The average chain molecular weight is approximately 9.7685 x 106 g/mol, that's around 93, 838 styrene monomers per average chain. The active sites of the enzymes secreted by the bacteria can only fit the free ends of the polystyrene chain, so out of a 93, 838 long chain of styrene monomers, only 0.002% of the chain is available to the enzyme.

Also, we considered the method the enzyme will use to find the end of the chain. It could either collide with the polymer chain suspended in solution, and at part of the chain hold on and shuffle along to the end - similar to restriction enzymes. The other method, with a chain suspended in solution, specifies that the enzyme can only start catalysing the reaction when it collides with a free end only; a much more lengthly, although possibly more realistic, senerio.

Michaelis-Menton equation

The Michaelis-Menton equation is a model for single-substrate reaction is used as a base for our model.

Where:
V0 is the initial veliocity of the reaction
Vmax is the rate of reaction when substrate is saturated
S is the substrate concentration
Km is the substrate concentration when the reaction rate is half the maximum and can be calculated using: k-1 + k2/k1

Resulting model

This model simulates enzymes that are released by the bacteria into the solution:

This model simulates a single bacteria in solution with the polystyrene substrate. Substrate concentration is 1M, where one monomer of Polystyrene is 104.1 g/mol. Also in this model, Tyro is the name of the enzyme digesting Polystyrene

.

In the model the polystyrene degrading enzyme is produced in the cell, and then transported by a specific transporter out of the cell and into the flask. Here it can work on the excess substrate polystyrene, catalysing its conversion to styrene.

This model is not an exact representation of the Psuedomonas behaviour. This is largely due to the fact the values for the M-M equation were gathered from a paper regarding Tyrosinase, but since there was no available data for Psuedomonas using Polystyrene we used this as a model. Further, it has been noted that Pseudomonas do not use free enzymes, but instead rely on the end of a polystyrene chain colliding with the active site on the bacteria [INSERT CITATION?].

However what this model can tell us, is the pattern of behaviour expected if Psuedomonas was modified to use free enzymes. This can be compared to results gained by observing the Psuedomonas at work, but sadly this data is still currently unavailable to us.

Enzyme modification

Programs, such as Pymol , allow one to manipulate the tertiary/quaternary structure of enzymes and other proteins, that have already had their structure determined by X-ray diffraction and/or NMR after they have been successfully crystallised. Whereas programs such as RASWIN only allow you to display these structures (in many different ways), Pymol takes this one stage further by giving one the power to manipulate individual residues to change the rotamer (direction/position the atoms in the residue) or even the residue itself (a kind of In silico mutagenesis). This means you can modify enzymes to such an extent that you may be able to fit any substrate you want into a particular enzyme's active site. However, in reality, the modified protein may adopt a completely different tertiary/quaternary structure which may cause it not to actually work as expected.

Polystyrene doesn't have its own degradation pathway due to the short time polystyrene has actually been in any great quantity in the world. Also, polystyrene is extremely unreactive, due to stable phenyl side chains present. Styrene, the polystyrene monomer has a known degradation pathway since being a much smaller molecule, it can diffuse into bacterial cells. The environment of the cell allows much more flexibility with proteins, as the highly reducing internal environment allows unstable complexes to persist (such as iron-sulphur complexes).

Firstly, we identified possible pathways that could be modified to accept polystyrene as a substrate. There were 2 potential pathways we could use: the styrene degradation pathway, and the toluene degrading pathway. Both have several different degradation pathways depending on the bacterial species/strain, but we ended up choosing the aerobic toluene degradation pathway from Pseudomonas putida, as the active sites of the pathway could remove/break the unreactive phenyl groups from the polystyrene chain.

Toluene degradation pathway in <i> P. putida</i>

The toluene degradation pathway. TDO is the enzyme we modified in silico using Pymol, and is Toluene 2,3-dioxygenase, which is made up of an alpha and beta subunit (TodC1 and TodC2) in a hexamer, with TodA and TodB acting as electron shuttles for the reaction from NADH to the enzyme. TodD is cis-1,2-dihydrobenzene-1,2-diol dehydrogenase which forms 3-methyl catechol. The other enzymes weren't focused on very much, though it was later found that there was no TodD crystal structure available online to modify. (George et. al, 2011)

Toluene 2,3-dioxygenase is the first enzyme of the pathway, and crystal structures are available online on the RCSB Protein Data bank. It was found that there was a gap directly into the active site, that if widened, could potentially fit polystyrene:

Toluene 2,3-dioxygenase is made up of alpha and beta subunits. The sticks show each non-hydrogen atom, and the dots show the area each atom takes up. The pink residues indicate the active site residues, and the orange molecule is toluene. As can be seen, there is only a small gap when the enzyme has toluene bound that links the active site to the external environment (from 3EN1). A interactive version of 3EN1 is available Here

However, there are a few residues that cause this gap to be so small, which we modified to amino acids with smaller sidechains, but that were still hydrophobic residues to widen the gap, potentially allowing polystyrene into the active site. The residues we modified were Met220→Ala220 (which when the Toluene 2,3-dioxygenase was blast searched, came up as a natural variation in some dioxygenases), Val421→Ala421, Tyr422→Leu422 and Tyr266→Val266, which created a bigger gap, which when the polystyrene structure was added to the active site and was sculpted, barely changed the active site structure as can be seen below:

Modified Toluene 2,3-dioxygenase enzyme with a similar view to the image above. Again, the pink residues are the active site residues. The white residues are those that have been modified. (left) The polystyrene substrate has been omitted for clarity, but note the gap is much bigger now (modified from 3EN1). (right) The polystyrene is present here: note the very tight fit into the gap

A side view of the modified enzyme. Note that the terminal phenyl group is in the active site

This view is just of the toluene 2,3-dioxygenase active site (blue), overlapped with the modified version (pink). Notice how similar the active site shape is, which means that potentially, the active site could still catalyse the dioxygenase reaction.

Whether or not the relatively large change in the positioning/angle of the polystyrene aromatic ring in the active site would hinder the reaction or not needs to be studied. Because there are also 2 proteins involved in transporting electrons to the enzyme, as few of the modified residues, if any are on the surface, so shouldn't affect binding. A future project could actually get this synthesised and assayed to see whether the modifications could allow polystyrene to bind in the active site, and cause hydroxylation of the side chain.

References

Guazzaroni, M., et al, A Novel Synthesis of Bioactive Catechols by Layer-by-Layer Immobilized Tyrosinase in an Organic Solvent Medium, ChemCatChem, 4, 89-99, 2012.