Part:BBa_K4179011
PT1a with the donor docking domain switched to the domain of PT1L
This sequence codes for a prenyl transferase - PT1a[1], with the donor docking domain switched to the domain of PT1L[1]. This enzyme should catalyze the formation of DMS from umbelliferone.
Origin
PT1a – Citrus limon PT1L – Humulus lupulus
Prenylation
Prenylation is a structural modification in which an isoprenoid moiety, prenyl being the most common, is transferred from a donor molecule to an acceptor. Prenylation of aromatic substrates causes the enhancement of their bioactivity which is a crucial step in the biosynthesis of biologically active secondary metabolites, which are important in the survival and disease resistance of many plant species. To perform prenylation, plants use membrane-bound aromatic prenyltransferases (PTs) that transfer isoprenoid moieties from pyrophosphate donor substrates to aromatic acceptor substrates [2].
Motivation for creating chimera enzyme
PT1a is a prenyltransferase that originated from Citrus limon and was expressed in yeast[2]. This prompted us to attempt expressing it in bacteria. This enzyme uses the desired acceptor but not the desired donor. PT1L is a prenyltransferase that uses the desired donor but not the desired acceptor. In this part, we switched the PT1a domain responsible for binding the donor molecule with the equivalent domain of PT1L.
Identifying the UbiA domain in each enzyme
We decided to identify the site that is responsible for the prenylation reaction and change it so it will use the correct donor. We hypothesized that this switch will enable the bacteria to express the enzyme while changing the donor molecule – giving our desired reaction. UbiA is a domain shared by a family of prenyltransferases – each catalyzing prenylation reaction[3]. For that reason, we deduced that this is the domain we should target. We used the Pfam database[4] to find the amino acids positions consisting of the UbiA domain.
Identifying donor docking domain
We then turned to identify the specific site responsible for donor docking. We found 2 other enzymes that were expressed in yeast[5,6] and uses the correct donor (with the wrong substrate) – G4DT originated from Humulus lupulus[1] and PT1L originated from Glycine max[1]. We again located the UbiA domains and used blast[7] to compare the 2 sequences expecting to find homology (because of the shared donor). We found a site consisting of 16 amino acids having high homology. We also noticed that the motif we found has 2 aspartates in the middle of it. We further noticed the presence of aspartates in other UbiA domains in other enzymes. It looked significant so we reviewed the literature and found out that aspartates are crucial for the action of the prenyltransferase as they interact with Mg2+ which is a cofactor for the enzyme[8]. All of the above led us to believe we identified the motif responsible for binding the desired donor (dimethylallyl diphosphate). To identify the site in the lemon enzyme responsible for binding the geranyl diphosphate we used blast to compare the UbiA of PT1a and (PcPT). We saw several homologies but searched for a sequence containing aspartates. We found a smaller homologous sequence – which we predicted because the two enzymes have different donors. We then assumed that the motif size should be 16 amino acids long and identified the site to be the homologous part (10 amino acids) plus the upstream 6 amino acids.
Domain switch
We switched between the identified sites to create a chimera enzyme. We validated the folding of our enzymes using Phyre2[9] and tested the chimeras' capability to bind dimethylallyl diphosphate using Dockthor[10]. The results showed that our chimeras bind the prenyl group even better than the original enzyme bind the geranyl group.
The team of Technion 2022 used this part in a construct (BBa_K4179013) designed for the purpose of introducing decursin’s biosynthetic pathway into E. coli. The team’s starting point was umbelliferone, which had to be prenylated to yield DMS. and thus, this enzyme was needed to perform the first reaction in the pathway.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 403
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1213
Illegal PstI site found at 403 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 403
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 403
Illegal NgoMIV site found at 818 - 1000COMPATIBLE WITH RFC[1000]
This part includes the gene of ChimeraI, in addition to two restriction sites, one at every end of the gene. NdeI restriction site was added to the N-terminus side of the sequence, directly before the methionine codon of the ChimeraI gene. NheI restriction site was added to the C-terminus side of the sequence, directly after the end of the gene.
A stop codon is not present at the end of the ChimeraI gene, due to the gene being cloned upstream to a P2A (BBa_K4179005)-mCherry(BBa_K106005)sequence. In this genetic system (BBa_K4179013) , the mCherry expression is tied directly to the start codon of ChimeraI, while not being covalently bound to it. In such a design the mCherry signal corresponds to a 1:1 ratio, at the very least, which allows for a lower estimate of ChimeraI’s expression.
References
1. Bateman, Alex, et al. “Uniprot: The Universal Protein Knowledgebase in 2021.” Nucleic Acids Research, vol. 49, no. D1, 2020, https://doi.org/10.1093/nar/gkaa1100.
2. Munakata, Ryosuke, et al. “Molecular Cloning and Characterization of a Geranyl Diphosphate-Specific Aromatic Prenyltransferase from Lemon .” Plant Physiology, vol. 166, no. 1, 2014, pp. 80–90., https://doi.org/10.1104/pp.114.246892.
3. Li, Weikai. “Bringing Bioactive Compounds into Membranes: The UbiA Superfamily of Intramembrane Aromatic Prenyltransferases.” Trends in Biochemical Sciences, vol. 41, no. 4, 2016, pp. 356–370., https://doi.org/10.1016/j.tibs.2016.01.007.
4. Mistry, Jaina, et al. “Pfam: The Protein Families Database in 2021.” Nucleic Acids Research, vol. 49, no. D1, 2020, https://doi.org/10.1093/nar/gkaa913.
5. Akashi, Tomoyoshi, et al. “Molecular Cloning and Characterization of a cDNA for Pterocarpan 4-Dimethylallyltransferase Catalyzing the Key Prenylation Step in the Biosynthesis of Glyceollin, a Soybean Phytoalexin .” Plant Physiology, vol. 149, no. 2, 2008, pp. 683–693., https://doi.org/10.1104/pp.108.123679.
6. Li, Haoxun, et al. “A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway.” Plant Physiology, vol. 167, no. 3, 2015, pp. 650–659., https://doi.org/10.1104/pp.114.253682.
7. Altschul, Stephen F., et al. “Basic Local Alignment Search Tool.” Journal of Molecular Biology, vol. 215, no. 3, 1990, pp. 403–410., https://doi.org/10.1016/s0022-2836(05)80360-2.
8. Bräuer, Lars, et al. “A Structural Model of the Membrane-Bound Aromatic Prenyltransferase UbiA Frome. Coli.” ChemBioChem, vol. 9, no. 6, 2008, pp. 982–992., https://doi.org/10.1002/cbic.200700575.
9. Kelley, Lawrence A, et al. “The PHYRE2 Web Portal for Protein Modeling, Prediction and Analysis.” Nature Protocols, vol. 10, no. 6, 2015, pp. 845–858., https://doi.org/10.1038/nprot.2015.053.
10. Guedes, Isabella A., et al. “New Machine Learning and Physics-Based Scoring Functions for Drug Discovery.” Scientific Reports, vol. 11, no. 1, 2021, https://doi.org/10.1038/s41598-021-82410-1.
11. Pettersen, Eric F., et al. “UCSF Chimerax: Structure Visualization for Researchers, Educators, and Developers.” Protein Science, vol. 30, no. 1, 2020, pp. 70–82., https://doi.org/10.1002/pro.3943.
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