Team:Tuebingen/Results/Modeling

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     <h1> Protein modelling with &alpha;-<i>N</i>-acetylgalactosaminase from <i> E. meningoseptica </i></h1>
     <h1> Protein modelling with &alpha;-<i>N</i>-acetylgalactosaminase from <i> E. meningoseptica </i></h1>
      
      
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<p>Our aim was to improve the activity of &alpha; - N - acetylgalactosaminase from <i> E. meningoseptica </i>. There are many reasons for that: For instance, time for a conversion has to be fast for routine tasks in hospitals. An another example would be the enzyme’s loss of activity being fixed onto a membrane. In order to achieve this we used the experimentally - determined structure of &alpha; - N - acetylgalactosaminase from <a href="http://www.nature.com/nbt/journal/v25/n4/abs/nbt1298.html">Liu et al. (2007)</a>. </p>
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<p>Our modelling aims to improve the activity of &alpha; - N - acetylgalactosaminidase from <i> E. meningoseptica </i>. There would be many practical advantages for a more efficient enzyme: For instance, conversion has to be completed as fast as possible for routine tasks in hospitals. An another example would be to compensate for a possible loss of activity, by being fixed onto a membrane. In order to achieve this we used the experimentally - determined structure of &alpha; - N - acetylgalactosaminidase from <a href=”http://www.nature.com/nbt/journal/v25/n4/abs/nbt1298.html”>Liu et al. (2007)</a>. </p>
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<p>As described in “<a href="http://www.nature.com/nbt/journal/v25/n4/abs/nbt1298.html">Bacterial glycosidases for the production of universal red blood cells</a>”, the active site consists of Tyr307, Tyr225, His228, Glu149, Tyr179 and Arg213. With this we tried to examine the interactions of the residues with the substrate. The precise distances, which would allow interactions, is listed in Table 1. The values refer to Schulz & Schirmer’s  „<a href="http://www.springer.com/life+sciences/biochemistry+%26+biophysics/book/978-0-387-90334-7">Principles of Protein Structure</a>“. </p>
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<p>As described in “Bacterial glycosidases for the production of universal red blood cells”, the active site consists of Tyr307, Tyr225, His228, Glu149, Tyr179 and Arg213. With this we tried to examine the interactions of the residues with the substrate. The precise distances, which would allow interactions, are listed in Table 1. The values refer to R.H Schirmer’s  „Principles of Protein Structure“ [2]. </p>
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<img src="https://static.igem.org/mediawiki/2014/2/2f/Tue2014_Results_Modeling_Table_1_New_Different_distances_between_residues_which_would_allow_interactions.jpg">
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<p id="picText">Table 1: Different distances between residues which would allow interactions.</p>
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<p>It was found that the substrate in the active site (Figure 1) is not stabilized at OH - 6 by residues (http://www.rcsb.org/pdb/ligand/ligandsummary.do?hetId=A2G&sid=2IXB), as already mentioned in <a href="http://www.nature.com/nbt/journal/v25/n4/abs/nbt1298.html">Liu et. al. (2007)</a>. It has been verified with the Protein Interactions Calculator that no interactions with OH - 6 were detected. After that, we discussed with Prof. Dr. Thilo Stehle (Interfaculty Institute of Biochemistry) and decided that it would be possible to facilitate the nucleophile attack of the hydroxyl group from Tyr179. Furthermore we tried different mutations in Leu183 and Val186. We found that a mutation of Val186 to Asp186 should stabilize the substrate. The distance between OH - 6 and Asp186 - COOH is 2,9 Å and would be suitable for a hydrogen bond (Figure 2). Also the distance to other residues is over 5 Å. It is important to note that such negative charge could have a negative effect on the activity. Due to that we tried Asn186 (Figure 3), but the distance with 3,03 Å is not optimal. </p>
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<p>It was found that the substrate in the active site (Figure 1) is not stabilized at OH - 6 by residues <li><a href="http://www.rcsb.org/pdb/ligand/ligandsummary.do?hetId=A2G&sid=2IXB">(Substate Structure) </a></li>, as already mentioned in <a href=”http://www.nature.com/nbt/journal/v25/n4/abs/nbt1298.html”>Liu et. al. (2007)</a>. It has been verified with the Protein Interactions Calculator that no interactions with OH - 6 were detected.
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<p>We discussed again with Prof. Dr. Stehle, he made the point that we have no good prediction tool. We tried to simulate the binding in the active site with the mutation in AutoDock, but we did not get a realistic result. He also mentioned that it is very difficult to predict the activity in the mutated protein, it might strongly bind the substrate with the new hydrogen bond.</p>
 
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<p>In conclusion, the best option is a mutation of Val186 to Asp186 where no collisions with other residues occure and a stabilizing effect at OH - 6 is obtained. However, there is no statement about an effect to the activity of the active site. </p>
 
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<img src="https://static.igem.org/mediawiki/2014/d/df/Tue2014_Results_Modeling_Figure_1_New_Active_site_of_N-Agal_with_CASTp_in_PyMOL.png">
 
<img src="https://static.igem.org/mediawiki/2014/7/7b/Tue2014_Active_site_N-Agal4.png">
<img src="https://static.igem.org/mediawiki/2014/7/7b/Tue2014_Active_site_N-Agal4.png">
<p id="picText">Figure 1: Active site of N-Agal with CASTp in PyMOL.</p>
<p id="picText">Figure 1: Active site of N-Agal with CASTp in PyMOL.</p>
<p> </p>
<p> </p>
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<img src="https://static.igem.org/mediawiki/2014/2/2f/Tue2014_Results_Modeling_Table_1_New_Different_distances_between_residues_which_would_allow_interactions.jpg">
 
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<p id="picText">Table 1: Different distances between residues which would allow interactions.</p>
 
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<img src="https://static.igem.org/mediawiki/2014/5/59/Tue2014_Results_Modeling_Figure_2_mutationAsp186.jpg">
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<p id="picText">Figure 2: Mutation Asp186.</p>
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<img src="https://static.igem.org/mediawiki/2014/8/81/Tue2014_Results_Modeling_Figure_3_mutationAsn186.jpg">
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<p id="picText">Figure 2: Mutation Asn186.</p>
    
    
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Revision as of 21:58, 17 October 2014


Protein modelling with α-N-acetylgalactosaminase from E. meningoseptica

Our modelling aims to improve the activity of α - N - acetylgalactosaminidase from E. meningoseptica . There would be many practical advantages for a more efficient enzyme: For instance, conversion has to be completed as fast as possible for routine tasks in hospitals. An another example would be to compensate for a possible loss of activity, by being fixed onto a membrane. In order to achieve this we used the experimentally - determined structure of α - N - acetylgalactosaminidase from Liu et al. (2007).

As described in “Bacterial glycosidases for the production of universal red blood cells”, the active site consists of Tyr307, Tyr225, His228, Glu149, Tyr179 and Arg213. With this we tried to examine the interactions of the residues with the substrate. The precise distances, which would allow interactions, are listed in Table 1. The values refer to R.H Schirmer’s „Principles of Protein Structure“ [2].

Table 1: Different distances between residues which would allow interactions.

It was found that the substrate in the active site (Figure 1) is not stabilized at OH - 6 by residues

  • (Substate Structure)
    • , as already mentioned in Liu et. al. (2007). It has been verified with the Protein Interactions Calculator that no interactions with OH - 6 were detected.

    Figure 1: Active site of N-Agal with CASTp in PyMOL.

    Figure 2: Mutation Asp186.

    Figure 2: Mutation Asn186.

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