"
Team:Kent/modelling
From 2014.igem.org
(Created page with "<html> <head> <meta charset="utf-8"> <title>Univeristy of Kent iGEM </title> <style type="text/css"> </style> <link rel="stylesheet" href="https://2014.igem.org/Team:Kent/CSS?act...") |
|||
| (One intermediate revision not shown) | |||
| Line 31: | Line 31: | ||
<!-- end .sidebar1 --></div> | <!-- end .sidebar1 --></div> | ||
| - | <div class="content" | + | <div class="content"> |
| - | + | ||
| - | + | ||
<table width="950" border="0"> | <table width="950" border="0"> | ||
<tr> | <tr> | ||
| Line 57: | Line 55: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td><em>Figure 1: Three protein models superimposed. They are based on the same template from PDB</em></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td><div align="justify">Consurf [4,5] was used to identify sequence conserved residues amongst all the limonene synthase genes available in the protein data bank (PDB). The darker shades of pink highlight the location of residues which are highly conserved. White residues have an average level of conservation (similar or not completely different functional groups in residues) and the blue residues show variable regions. This highlights areas of functional importance in the enzymes. Limonene synthase was used as a model due to lack of sequence information regarding the other synthases. A large number of sequences are required to provide data with a higher confidence level. 61 different limonene synthase genes were used to generate this map.</div></td> | + | <td><div align="justify"> |
| + | <p> </p> | ||
| + | <p>Consurf [4,5] was used to identify sequence conserved residues amongst all the limonene synthase genes available in the protein data bank (PDB). The darker shades of pink highlight the location of residues which are highly conserved. White residues have an average level of conservation (similar or not completely different functional groups in residues) and the blue residues show variable regions. This highlights areas of functional importance in the enzymes. Limonene synthase was used as a model due to lack of sequence information regarding the other synthases. A large number of sequences are required to provide data with a higher confidence level. 61 different limonene synthase genes were used to generate this map.</p> | ||
| + | </div></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 69: | Line 70: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td><em>Figure 2: Model of limonene synthase showing sequence conservation amongst the 61 variants in the PDB. Blue (variable), white (average) and pink (conserved).</em></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td><p align="justify">This model suggests there are several domains important to the function of terpene synthases. The substrate-binding active site is of critical importance and therefore the residues around the site which are important in function are likely to be conserved.</p></td> | + | <td><p align="justify"> </p> |
| + | <p align="justify">This model suggests there are several domains important to the function of terpene synthases. The substrate-binding active site is of critical importance and therefore the residues around the site which are important in function are likely to be conserved.</p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 81: | Line 83: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td><em>Figure 3: Model of limonene synthase showing the metal-binding domain (red).</em></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td><p align="justify">The red highlighted region of the protein shows the DDXXD motif common to all terpene synthases. It is the metal binding domain responsible for binding Mg2+ or Mn2+ ions as co-factors for enzyme activity. <br> | + | <td><p align="justify"> </p> |
| + | <p align="justify">The red highlighted region of the protein shows the DDXXD motif common to all terpene synthases. It is the metal binding domain responsible for binding Mg2+ or Mn2+ ions as co-factors for enzyme activity. <br> | ||
An understanding of the functional regions of the synthases could allow the potential for site-directed mutagenesis to increase the activity of an enzyme. Altering residues could increase the Vmax values and or increase the affinity of the enzyme for its substrate. It could also increase the specificity of products from the reaction. For example zingiberene synthase produces low amounts of other compounds such as β-sequiphellandrene and α-bergamotene [6]. Mutagenesis at specific residues may increase the ratio of desired product and make the extraction of our desired product a simpler task.</p> | An understanding of the functional regions of the synthases could allow the potential for site-directed mutagenesis to increase the activity of an enzyme. Altering residues could increase the Vmax values and or increase the affinity of the enzyme for its substrate. It could also increase the specificity of products from the reaction. For example zingiberene synthase produces low amounts of other compounds such as β-sequiphellandrene and α-bergamotene [6]. Mutagenesis at specific residues may increase the ratio of desired product and make the extraction of our desired product a simpler task.</p> | ||
<p align="justify"> </p> | <p align="justify"> </p> | ||
| Line 102: | Line 105: | ||
<!-- end .content --></div> | <!-- end .content --></div> | ||
| + | |||
<div class="footer"> | <div class="footer"> | ||
Latest revision as of 01:03, 18 October 2014
 |
 |
 |
 |
 |
 |
Modelling |
This is the template structure to which zingiberene synthase, R-linalool synthase and limonene synthase were modelled. The models were made using Phyre2[1] based on the amino acid sequences available from uniprot [2]. The genes used in this project have been curated (Swissprot) so are more reliable than hypothetical proteins identified from homology. The colours show the overlap between the 3 sequences. This suggests that despite having very low sequence identity and producing different groups of terpenes, they are based on the same structure and therefore will have a similar mechanism of action. |
 |
 |
| Figure 1: Three protein models superimposed. They are based on the same template from PDB |
Consurf [4,5] was used to identify sequence conserved residues amongst all the limonene synthase genes available in the protein data bank (PDB). The darker shades of pink highlight the location of residues which are highly conserved. White residues have an average level of conservation (similar or not completely different functional groups in residues) and the blue residues show variable regions. This highlights areas of functional importance in the enzymes. Limonene synthase was used as a model due to lack of sequence information regarding the other synthases. A large number of sequences are required to provide data with a higher confidence level. 61 different limonene synthase genes were used to generate this map. |
 |
| Figure 2: Model of limonene synthase showing sequence conservation amongst the 61 variants in the PDB. Blue (variable), white (average) and pink (conserved). |
This model suggests there are several domains important to the function of terpene synthases. The substrate-binding active site is of critical importance and therefore the residues around the site which are important in function are likely to be conserved. |
 |
| Figure 3: Model of limonene synthase showing the metal-binding domain (red). |
The red highlighted region of the protein shows the DDXXD motif common to all terpene synthases. It is the metal binding domain responsible for binding Mg2+ or Mn2+ ions as co-factors for enzyme activity.
|
