A: General

Login at 3dmjs.bio-prodict.nl with your 3DM account. If you don’t have a 3DM account you can request one via the “get 3DM” tab. To be able to do this course you need at least a course login. After you have requested an account you can request a course login by sending an email to Joosten@bio-prodict.nl

After entering the login details you will see a 'Select 3DM system' page. Click on the 'Public 3DM Systems' checkbox and search for the “Phosphoenolpyruvate mutase/Isocitrate lyase” database. Open this 3DM system.

At the starting page of each 3DM database you see the 3DM data cycle. The icons in the circle represent links to the most important 3DM options.


Fungi can be pathogenic to plants and animals. It is known that the secretion of oxalate by these fungi is a commonly used strategy for their pathogenicity. The oxalate is toxic and can easily form crystals which demolish the cell wall of the host. The oxalate is produced from oxaloacetate catalyzed by the enzyme oxaloacetate hydrolase (OAH). This is the reaction:

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Fig 1. Reaction mechanism that produces oxalate.

We have generated a 3DM for the corresponding protein family. OAH falls in the Phosphoenolpyruvate mutase/Isocitrate lyase superfamily.

The OAH of niger is the best characterized OAH protein. This is the sequence:


For each protein in the 3DM database there is a “protein information” page that contains more detailed information.


3DM offers several ways to select a subset of sequences. Once a subset is selected a mini 3DM can be generated for this subset. All 3DM functionalities, such as the correlated mutations, are regenerated and can separately be analyzed. The data of a subset can also be compared to the data of the full set of sequences or with other previously defined subsets.

With the search option we have made a subset called "OXALATE PRODUCERS" that contains the proteins available in this 3DM system for fungi of which it is known that they can produce oxalate:

				Aspergillus clavatus
				Neosartorya fischeri
				Penicillium chrysogenum
				Penicillium marneffei
				Talaromyces stipitatus
				Sclerotinia sclerotiorum
				Aspergillus niger
				Sclerotium cepivorum
				Aspergillus terreus
				Aspergillus fumigatus
				Botryotinia fuckeliana

At the alignment statistics pages change between the full dataset and the "OXALATE PRODUCERS" you just made using the 'Subset' menu in the header on top of 3DM and see how the graphs change.

3DM always generates an extra histogram for each subset that shows the residues that are specifically conserved in the selected subset (the histogram called subset specific conservation). The highest scoring residues are around 3D positions 157.

Important here is to realise that these are positions that are not just simply conserved in this subset of oxalate producing fungi, but the corresponding residues are absent from the rest of the sequences in the superfamily. In other words these residues are specific for the subset.

On the alignment page:

Take home message: the data you are looking at is always depending on the subset tab that is selected.

Click on the correlated mutation menu item. Make sure you have the full data set tab active.

Correlated mutations calculated for a superfamily alignment often reflect positions important for specificity because superfamily alignments contain enzymes with different specificities (do you understand this concept?)

The “Top Correlation Heatmap” page shows the alignment positions of which the residues mutate simultaneously (definition of a correlated mutation).

Go back to the alignment statistics page.

Homology modeling of OAH niger with its substrate oxaloacetate and the design of an inhibitor

Build a homology model.

3DM selected three structures as potential good templates. In this course you will learn how to select the best template, make the best alignments, etc., but for now we will use 3LYEA -> it does have the best resolution (e.g. quality).

By now you should have the model in Yasara.

Load an inhibitor

Structures can be loaded directly in Yasara from the 3DM database via the 3DM -> load from 3DM option. Loading structure files via the 3DM menu ensures that the structures are all superimposed, co-crystalized compounds will have be positioned in the active site and proteins will have the 3D numbering.

Build oxaloacetate from this inhibitor.

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Fig 2. Structure of oxaloacetate.

This is the structure of oxaloacetate. We are very lucky since it is very similar to the structure of the 1M1BA inhibitor. Simply swapping the SO3 group with a CO2 group will do the job.

The reaction mechanism of isocitrate lyase (ICL) is known for quite a while (fig 3). In this reaction mechanism the H of the blue OH group donates an electron, makes a double bond, and splits of the COOH group.

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Fig 3. Reaction mechanism of ICL (above) and the structure of oxaloacetate (below)

Actually, oxaloacetate in water is in equilibrium with its diol form (figure 4).

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Fig 4. Oxaloacatate is in equilibrium with its diol.

Until today OAH is the only known enzyme of this superfamily that has a substrate in a diol form. So the extra OH is unique to OAH.

Modeling the extra OH in the active site with the “swap” option does not work, because yasara can’t deal with changing the double bond of C=O to the single bond of C-OH without proper EM (try to make the diol with the swap option if you like).

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Fig 5. The result of energy minimization performed on the diol form of oxaloacetate in the OAH model.

In 2008 a model of OAH was generated similar to the way you did it today. With this model we were already in 2008 able to:

  1. Reveal the OAH specific serine 157 (figure 6)
  2. Reveal the reaction mechanism of OAH (via the diol substrate)
  3. Show the relation between oxalate production and pathogenicity of fungi
  4. Make a very strong inhibitor of OAH (potential anti-fungal drug)

The inhibitor was designed by organic chemists that realized they had to make a compound that is 100% in the diol form. This was the case with difluoro-oxaloaceate. This compound indeed proved to be a very strong inhibitor of OAH and was later crystalized together with OAH of the fungus Cryphonectria Parasitica (pdb file 3M0JA).

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Fig 6. Picture of the model of OAH taken from the 2008 publication: Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker-based method. This picture clearly shows the predicted Ser157 H-bridge with the diol of oxaloacetate.

Extra questions

Position 157 is the center of the correlated mutation network. P is the most common residue at position 157 (is that correct?). We have generated a subset of sequences that have a P at position 157 called "P157"

Take home message: The function underlying correlated mutations heavily depend on the input alignment. Always look for additional data (in this case protein-protein interaction data -> did you find that?) that might explain a correlated mutation network.

The correlated mutations in this superfamily seem to reflect positions important for specificity. You want to change the specificity of OAH and you decide to rationally design a mutant library. Your screening method allows you to screen up to a 1000 mutant clones.