Laccase modeling

Laccases are enzymes that pair the oxidation of the substrate (microplastics) with the reduction of an OH to water. They are formed by 2 copper centers : The oxidation of the substrate takes place at the mononuclear center while the reduction of OH is performed at the trinuclear center. The dimensions of the protein are 70*50*20 Angstrom and its usual substrate ABTS is much smaller, if we want to use laccase to degrade by oxidation our microplastics we are faced with a problem of scale as we can see below :

Our goal is then to make the oxidation site more accessible to microplastics by modeling diverse designs whose structures were predicted with AlphaFold collab version 2 then evaluated in Chimera against a reference structure, the process of the modeling is mentioned right below.

Process :
1. Study of the structure of the laccases based on articles and annotate the protein sequences to highlight the different domains and amino acids that bind copper, see Figure 1 and 2. Visualizing the 3D structure on chimera to see how different secondary are connected to each other and what changes could be made so that the active site is more accessible to microplastics.

Figure 1 : Laccase 1 · Bacillus subtilis (strain 168) · EC:

Figure 2 : Laccase 2 · Trametes versicolor · EC:

2. Learning how to use chimera and the different tools it offers.
3. Target plausible modification possible based on the structure and information on it that we could find as mentioned above then make the corresponding prediction with Alpha Fold.
4. Once the prediction is done, if the LDDT score is superior to 90, the prediction is expected to be of high accuracy, between 70 and 90 the prediction for the backbone of the protein is considered good, less than that the model should be treated with caution. The file of the prediction is downloaded if the LDDT score is good (>70) and studied in chimera along the bacterial or fungal laccase.
5. Once chimera you can proceed with :

Table 1 : Alignment of reference structure and model
Model B1
Sequence :

In gray : reference structure, in pink the model
Model B2
Sequence :
Model B3
Sequence :
Model F1
Sequence :

In gray : reference structure, in green the model
Model F2
Sequence :

Table 2 : Result of the measurement of the distance between the oxidation site and the microplastic, the global and specific RMSd of the copper-binding amino acids
Organisms Source Model predicted with alpha Microplastic’s distance from oxidation site Global RMSd RMSd of specific amino acid
Bacillus subtilis Wild Type (as a control of the different measure) 13.4 Å /
B1 : deletion of residu 211-223 / 320-330 7.429 Å 1.053 Å 0.209 Å
B2 : deletion of residu 211-223 / 320-330 and ALA replaced by PRO 8.759 Å 1.069 Å 0.204 Å
B3 : deletion of residu 211-223 / 320-330 / boucle couvercle and ALA375 replaced by PRO 5.863 Å 1.020 Å >2 Å
Trametes versicolor Wild Type (as a control of the different measure) 8.669 Å /
F1 : deletion of residu 154 à 165 / 264 à 274 6.938 Å 0.733 Å Å
F2: deletion of residu 154 à 165 / 264 à 274 mutation ALA 329 par PRO 6.098 Å 0.744 Å 0.200 Å

The different results presented in table 2 allowed us to select the model we would like to produce. The distance between the oxidation site and the microplastic is better for model B3 but the RMSd obtained for the specific residues is higher than 2 angstroms showing an important divergence between the reference structure and model 3 which could lead to the total loss of the activity. Model B2 has a good global and local RMSd but the distance between the oxidation site and the microplastic is slightly higher than that of model B1, so we decided after exchanging also with our Primary PI to order the sequence of model B1 in order to launch the production. Finally we could not test the production of a fungal model because the model was finalized a little too late. We would not have had enough time to test its production and its activity.

References :