Revision as of 22:27, 11 October 2022

Arabidopsis thaliana 4-Coumarate:CoA Ligase Mutant (At4CL L57I/L460H)

Background

The Arabidopsis thaliana 4-coumarate:CoA Ligase (At4CL) is an enzyme that catalyses the activation of 4-coumaric acid with Coenzyme A to form 4-coumaroyl-CoA.

4CL is a key enzyme of the general phenylpropanoid pathway that has been used for the production of multiple polyphenols including naringenin, resveratrol, and pterostilbene. This version of the enzyme is a mutant with amino acid changes L57I and L460H. These mutations have been shown to increase catalytic efficiency from 6.72 (wild-type) to 31.76 μmol/h/g (Yan et al., 2021).

The ORF of this compound was obtained from the iGEM registry: BBa_K1033001. This sequence was then codon optimized for E. coli K12, mutated at the desired positions, and two stop codons (TAATAA) were added to terminate translation.

Usage and Biology

Sequence and Features

Functional Parameters

This new part was made as an improvement upon BBa_K1033001, as detailed below. The part BBa_K801093 has the same protein sequence as BBa_K1033001, but has previously been used in yeast.

Molecular Docking

Introduction

As part of engineering our plasmid constructs to include the genes for four enzymes in the pathway to synthesize pterostilbene, ensuring the most efficient enzymes possible was important. The four genes we decided to use for our biosynthetic pathway for pterostilbene production were mutant versions of the wild type forms of the enzymes RgTAL, At4Cl, VvSTS and VvROMT found by (Yan et. al, 2021). These mutations claimed to increase the pterostilbene production titre by a factor of 13.7 compared to their respective wild type forms. As this specific mutant At4CL1 was found in literature to have greater catalytic efficiency than the Wild-type (Yan et. al, 2021), it presented a potential variant to use in our plasmid constructs. As the wild type form of At4CL was already present in the iGEM registry under the code BBa_K1033001, created by iGEM13_Uppsala, we decided to explore experimental avenues that would support and further characterize this wild type and mutant variant (BBak4388002) to justify our reasoning for choosing the mutant form of the enzymes and to improve the part. We aimed to determine differences in binding energy and affinity between this mutant and its wild type computationally to further investigate and characterise the mutant and wild type for use in our project.

At4CL

4-Coumaroyl-CoA Ligase (4CL) catalyses the conversion of p-Coumaric acid to p-Coumaroyl-CoA. The BioBrick At4CL1 part BBa_K1033001 has 100% local identity with Genbank Accession number AAA82888.1, and Uniprot accession code Q42524.

The mutant At4CL1 found to have greater catalytic efficiency than the Wild-type has point mutations at L57I and L460H.(Yan et. al, 2021) Docking simulations were performed to obtain KD and ΔG of both the wild-type and mutant.

Obtaining PDB models for the Wild Type and Mutant

The closest template found from the Swiss Model was part of a fusion protein with Stilbene Synthase from (Wang et. al, 2011). Structural differences of At4CL1 as part of the fusion protein were reported to not vary drastically when compared with At4CL1 alone,(Wang et. al, 2011) and the Root Mean Squared Deviation (RMSD) value comparing the 4CL section of the 3TSY fusion protein to the At4CL1 Alphafold model was low (0.445), suggesting the Alphafold model is similar in structure with the 3TSY model, as can be seen in Figure 1. As we were investigating whether point mutations in only two locations had an effect on binding energy, obtaining the most accurate PDB model was important. Using a model of a homolog would not have been accurate enough for docking simulations, given that catalytic specificity and efficiency for a specific substrate can vary significantly even between the different At4CL isoforms. The Alphafold model was chosen as it was of high confidence, and had a more complete structure than 3TSY could provide.(Figure 1 and 2) The mutant At4CL1 PDB was created by mutagenesis in Pymol of the Wild-type Alphafold model to introduce the mutations L57I and L460H. Our new part, BBak4388002, contains these mutations.

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Figure 1. Alphafold model (purple), 4CL section from fusion protein 3TSY (blue). RMSD = 0.445. Made using Pymol. The Alphafold structure can be seen to be more complete than the 4CL section from the fusion protein 3TSY.

Figure 2. Alphafold model of the Wild-type 4-Coumaroyl-CoA Ligase from Arabidopsis thaliana. The legend indicates levels of confidence in structural accuracy. pLDDT is a per-residue metric of the structure’s confidence on a scale of 0 - 100.

Choosing a Docking programme

Predicted docking energies from both Autodock Vina or Autodock4 have been found to correlate well with experimentally determined docking energies, with values obtained using Autodock4 consistently closer to experimentally determined values. Autodock4 has been found to be the superior option for estimating binding affinity (Nguyen et. al, 2020), making it the preferred option for our purposes. Yasara Structure was therefore used to perform docking simulations using Autodock4.

Using Yasara

Energy minimisation was run in Yasara for both enzyme and substrate to find the most energetically favourable conformations. The PDB structures for the enzymes were found to have improved Molprobity results after Energy minimisation compared to before. Each Autodock4 docking simulation performed 25 runs, and clustered results with high similarity into distinct complex conformations. The results from the best-scoring distinct complex conformation for wild type and mutant according to Autodock4 was selected for comparison.

Results and Conclusion

The value of KD found for the mutant At4CL1 was lower than that of the wild type, suggesting it has better affinity with p-Coumaric acid. (Table 1) This may help explain the greater catalytic efficiencies of the mutant variant compared to its wild type.(Yan et. al, 2021) The At4CL mutant had a more favorable binding energy change with p-Coumaric acid than the wild-type.

Table 1. Results of the top distinct complex conformation in each of the docking simulations run with Autodock4 (AD4) through Yasara Structure. These were performed for both the wild type and mutant At4CL to obtain KD and ΔG.

Figure 3. Yasara docking simulation result with Autodock4. Shown is the Wild-type 4-Coumaroyl-CoA Ligase enzyme from Arabidopsis thaliana in complex with the substrate p-Coumaric acid. Yasara automatically colour codes the secondary structure elements as follows: Alpha helices (dark blue), inside of helix (grey), beta sheets (red), turn (light green), helix 310 (yellow), coil (light blue).

From the Yasara docking simulation, the results for wild type and mutant were compared in Pymol. The position of p-Coumaric acid when bound to wild type and mutant At4CL is different and can be seen in figure 4.

Further examination of the amino acid residues of At4CL involved in binding to p-Coumaric acid can be seen in Figure 5. As seen in Table 2, though most of the amino acid residues involved in binding to p-Coumaric acid are predicted to be the same in both mutant and wild type, some residues involved in the binding are different in the mutant and wild-type.

References

Wang, Y., Yi, H., Wang, M., Yu, O., & Jez, J. M. (2011). Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase. Journal of the American Chemical Society, 133(51), 20684–20687. https://doi.org/10.1021/ja2085993

Nguyen, N. T., Nguyen, T. H., Pham, T., Huy, N. T., Bay, M. V., Pham, M. Q., Nam, P. C., Vu, V. V., & Ngo, S. T. (2020). Autodock Vina Adopts More Accurate Binding Poses but Autodock4 Forms Better Binding Affinity. Journal of chemical information and modeling, 60(1), 204–211.. https://doi.org/10.1021/acs.jcim.9b00778

Yan, Z. B., Liang, J. L., Niu, F. X., Shen, Y. P., & Liu, J. Z. (2021). Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering. Frontiers in microbiology, 12, 710405. https://doi.org/10.3389/fmicb.2021.710405

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