Coding

Part:BBa_K801093

Designed by: Ingmar Polte   Group: iGEM12_TU_Munich   (2012-09-21)

4-coumarate--coenzyme A ligase (4CL) coding region

4-coumarate--coenzyme A ligase (4CL) coding region from Arabidopsis thaliana.

Background and priniciples

Structure of Xanthohumol

Usage and Biology

Inhibition of the metabolic activation of procarcinogens:

2-amino-3-methylimidazo[4,5-f]quinolone, found in cooked meat, verified as a procarcinogen in an ames salmonella mutagenicity test. The inhibition is probably a result of an inhibition of the cytochrome P 450 enzymes Cyp1A1, Cyp1B1 and Cyp1A2 (phase 1 enzymes). But in order to achieve a clear inhibition, plasma concentrations of 1 µM would be necessary. In a study with male rats oral administration of xanthohumol (50 mg/kg) led to concentration maximums of 65 -180 nM after 4 h. Improved resorption of xanthohumol could be a possible target for innovation [[http://www.ncbi.nlm.nih.gov/pubmed/11240137 Yilmazer et al. 2001a], [http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al. 2000b], [http://www.ncbi.nlm.nih.gov/pubmed/10752639 Henderson et al., 2000], [http://www.ncbi.nlm.nih.gov/pubmed/12481418 Gerhauser et al., 2002]].

Induction of carcinogen-detoxifying enzymes (phase 2 enzymes):

P450-activated carcinogens get conjugated to endogenous ligands (gluthathione, glucoronic acid, acetate and sulfate) by phase 2 enzymes to facilitate excretion. Therefore the induction of phase 2 enzymes should enhance the protection against carcinogenesis. Xanthohumol cat concentrations of 2.1-10.1 µM could induce quinone reductase (detoxification of quinones by conversion to hydroquinones which can be conjugated) in hepatoma Hepa 1c1c7 cells. It was shown that xanthohumol could selectively induce quinone reductase without causing a transcriptional activation of Cyp1A1 [[http://www.ncbi.nlm.nih.gov/pubmed/11038156 Miranda et al., 2000c], [http://www.ncbi.nlm.nih.gov/pubmed/12481418 Gerhauser et al., 2002]].

Inhibition of tumor growth at an early stage:

Xanthohumol showed an inhibition of the proliferation of breast cancer (MCF-7) and ovarian cancer (A-2780) in vitro at IC50 values of 13 and 0.52 µM http://www.ncbi.nlm.nih.gov/pubmed/10418944 Miranda ''et al.'', 1999. Furthermore xanthohumol can inhibit the endogenous prostaglandin synthesis through inhibition of cyclooxygenase (COX-1 and COX-2) with IC50 values of 17 and 42 µM. An increased prostaglandin production has been associated with the uncontrolled proliferation of tumor cells http://www.ncbi.nlm.nih.gov/pubmed/12481418 Gerhauser ''et al.'', 2002. Pharmacokinetic studies for xanthohumol based on beverages with an xanthohumol content of 50 mg/l in humans are part of actual research activities.

Antioxidant activities:

Xanthohumol at 5 µM decreased conjugated diene formation as a measure for lipid peroxidation by more than 70 % after 5 h of incubation in an in vitro assay (protection of LDL from Cu2+ induced oxidation). Furthermore xanthohumol was shown to scavenge hydroxyl-, peroxyl- and superoxide anion radicals http://www.ncbi.nlm.nih.gov/pubmed/11038156 Miranda ''et al.'', 2000c. </div>

Biosynthesis

Biosynthesis of Xanthohumol

The biosynthetic pathway of 4-coumaroyl-coenzyme A starts with the conversion of L-Phenylalanine to cinnamate, being catalyzed by phenylalanin ammonia lyase (PAL) [A]. PAL also shows activity in converting tyrosine to p-coumarate, but with a lower efficiency [B]. The cinnamate 4-hydroxylase (C4H) catalyzes the synthesis of p-hydroxycinnamate from cinnamate and 4-coumarate [C]: CoA ligase (4CL) converts p-coumarate to its coenzyme-A ester, activating it for reaction with malonyl CoA [D] [Trantas et al., 2009]. The flavonoid biosynthetic pathway starts with the condensation of one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, yielding naringenin chalcone. This reaction is carried out by the enzyme chalcone synthase (CHS) [E]. Chalcone is isomerised to a flavanone by the enzyme chalcone flavanone isomerase (CHI). From these central intermediates, the pathway diverges into several side branches, each resulting in a different class of flavonoids, such as xanthohumol.

There are 5 enzymes necessary for the biosynthesis of xanthohumol ([http://biocyc.org/META/NEW-IMAGE?type=NIL&object=PWY-5135 MetaCyc]):

Enzyme [A]: PAL = phenylalanine ammonia lyase: L-phenylalanin --> trans-cinnamate

Enzyme [D]: 4CL = 4-coumarate - coenzym A ligase: 4-coumarate --> 4-coumaroyl-CoA

Enzyme [E]: CHS = naringenin - chalcone synthase: 4-coumaroyl-CoA --> naringeninchalcone

Enzyme [F]: APT = aromatic prenyltransferase: naringeninchalcone --> desmethylxanthohumol

Enzyme [G]: OMT1 = chalcone O-methyltransferase: desmethylxanthohumol --> xanthohumol

Jiang et al succeeded in the biosynthesis of several flavonoids in Saccharomyces cerevisiae by the assembly of a plasmid containing three required enzymes (pKS2µHyg-PAL-4CL-CHS) and thereby showed the proof of principle. The activity of each enzyme was demonstrated and the presence of naringenin, which forms the product of the three enzymes( PAL, 4CL, CHS), was shown. http://www.ncbi.nlm.nih.gov/pubmed/14704995 Jiang and Morgan, 2004














Characterization

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1086
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1653
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1285


References


KCL iGEM 2022

This part was used in yeast, but for use in bacteria, an improved part has been made, as detailed below. BBa_K801093 has the same protein sequence as BBa_K1033001. The new part BBak4388002, containing mutations, has been shown by Yan et. al (2021) to have improved catalytic efficiency in E. coli, and we have further characterised it.

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.

improvement-figure-1.png"

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. improvement-figure-2.png

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.

table-1-improvement.png



improvement-figure-3.png

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.


improvement-figure-4.png

Figure 4. p-Coumaric acid (Yellow) in complex with wild-type At4CL (Light blue), overlaid onto p-Coumaric acid
(Red) in complex with mutant At4CL (Dark blue). Created in Pymol using Yasara docking simulation results.


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.


Table 2. At4CL amino acid residues involved in binding to p-Coumaric acid. Comparisons of amino acid residues from wild type and mutant At4CL from Autodock4 results using Yasara

improvement-table-2.png


improvement-figure-5.png

Figure 5. A) p-Coumaric acid (purple) in complex with the wild-type At4CL. B) p-Coumaric acid (Dark green) in complex with mutant At4CL. Created in Pymol.

 

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

Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S., Ballard, A. J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., Back, T., … Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596(7873), 583–589. https://doi.org/10.1038/s41586-021-03819-2

Varadi, M., Anyango, S., Deshpande, M., Nair, S., Natassia, C., Yordanova, G., Yuan, D., Stroe, O., Wood, G., Laydon, A., Žídek, A., Green, T., Tunyasuvunakool, K., Petersen, S., Jumper, J., Clancy, E., Green, R., Vora, A., Lutfi, M., Figurnov, M., … Velankar, S. (2022). AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic acids research, 50(D1), D439–D444. https://doi.org/10.1093/nar/gkab1061

Krieger, E., & Vriend, G. (2014). YASARA View - molecular graphics for all devices - from smartphones to workstations. Bioinformatics (Oxford, England), 30(20), 2981–2982. https://doi.org/10.1093/bioinformatics/btu426

The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

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Categories
//cds
//cds/biosynthesis
//chassis/eukaryote/yeast
Parameters
None