Difference between revisions of "Part:BBa K4179002"

 
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<partinfo>BBa_K4179002 short</partinfo>
 
<partinfo>BBa_K4179002 short</partinfo>
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[[Image:FcPT1 image.png|thumb|400px|'''Structure of FcPT1 enzyme [3]''']]
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[[Image:PsPT1 reaction.png|thumb|400px|'''Prenylation of umbelliferone using DMAPP as a prenyl donor''']]
  
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F. carica prenyltransferase 1 (FcPT1) belongs to the UbiA superfamily, a prenyltransferase (PT) family of membrane-bound proteins possessing two aspartate-rich motifs that are conserved motifs crucial for the divalent cation-dependent prenylation.
 
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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 is known to enhance their bioactivity and is an essential step in the biosynthesis of biologically active secondary metabolites, which play an integral role in the survival and disease resistance of many plant species. To perform prenylation, plants use membrane-bound aromatic PTs that transfer isoprenoid moieties from pyrophosphate donor substrates to aromatic acceptor substrates.
 
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FcPT1 is an umbelliferone dimethylallyltransferase (UDT), a PT that is specific for the first reaction step in the furanocoumarin (FC) biosynthetic pathway. It mediates a prenylation reaction in which umbelliferone is the acceptor of the prenyl group, and dimethylallylpyrophosphate (DMAPP) is the donor, producing DMS if the prenylation takes place at the C6 position of umbelliferone, and osthenol if prenylation occurres in the C8 position.
 
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To characterize the enzymatic function of FcPT1, its full CDS was transiently expressed in N. benthamiana by Munakata et al. Substrate specificity of FcPT1 for prenyl acceptors was evaluated with various aromatic compounds using DMAPP as a prenyl donor. Incubations with various simple coumarins and FCs showed that this enzyme recognized umbelliferone and 5- methoxy-7-hydroxycoumarin (5M7H) as prenyl acceptors. The specificity of FcPT1 for prenyl donor substrates was also assessed using geranyl diphosphate, farnesyl diphosphate and GGPP in the presence of umbelliferone or 5M7H, but no products were detected, suggesting that FcPT1 specifically transfers a dimethylallyl moiety to umbelliferone and 5M7H.
 
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TMHMM analysis predicted that the enzyme has multiple transmembrane alpha-helices and CHLOROP predicted that its N-terminal region has a transit peptide.
 
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Subcellular localization of FcPT1
 
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Using GFP fusion, it was found that the enzyme is localized to chloroplasts, which suggests that FcPT1 localizes to plastids, which is consistent with the synthesis of prenyl donors for UbiA PTs, including DMAPP, via the MEP pathway.
 
  
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F. carica prenyltransferase 1 (FcPT1) belongs to the UbiA superfamily, a prenyltransferase (PT) family of membrane-bound proteins possessing two aspartate-rich motifs that are conserved motifs crucial for the divalent cation-dependent prenylation [1].
  
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===Usage and Biology===
 
  
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===Prenylation===
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<span class='h3bb'>Sequence and Features</span>
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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].
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===Usage and biology===
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FcPT1 is an umbelliferone dimethylallyltransferase (UDT), a PT that is specific for the first reaction step in the furanocoumarin (FC) biosynthetic pathway. It mediates a prenylation reaction in which umbelliferone is the acceptor of the prenyl group, and dimethylallylpyrophosphate (DMAPP) is the donor, producing DMS if the prenylation takes place at the 6th carbon position of umbelliferone, and osthenol if prenylation occurres at the 8th carbon position.
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The team of Technion 2022 used this part in a construct [https://parts.igem.org/Part:BBa_K4179003 (BBa_K4179003)] 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.
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===Sequence and Features===
 
<partinfo>BBa_K4179002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4179002 SequenceAndFeatures</partinfo>
  
  
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This part includes the gene of FcPT1, 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 FcPT1 gene. NheI restriction site was added to the C-terminus side of the sequence, directly after the end of the gene.
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===Functional Parameters===
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<partinfo>BBa_K4179002 parameters</partinfo>
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A stop codon is not present at the end of the FcPT1 gene, due to the gene being cloned upstream to a P2A [https://parts.igem.org/Part:BBa_K4179005 (BBa_K4179005)]-mCherry [https://parts.igem.org/wiki/index.php?title=Part:BBa_K106005 (BBa_K106005)] sequence. In this genetic system [https://parts.igem.org/Part:BBa_K4179003 (BBa_K4179003)] , the mCherry expression is tied directly to the start codon of FcPT1, 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 FcPT1’s expression.
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==References==
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1. Munakata R, Kitajima S, Nuttens A et al. Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants. New Phytologist 2020; 225: 2166–2182.
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2. de Bruijn WJC, Levisson M, Beekwilder J, van Berkel WJH, Vincken J-P. Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories. 2020;
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3. Bateman A, Martin MJ, Orchard S et al. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 2021; 49: D480–D489.

Revision as of 15:40, 5 October 2022


Ficus caricia prenyl transferase (FcPT1)

Structure of FcPT1 enzyme [3]
Prenylation of umbelliferone using DMAPP as a prenyl donor


F. carica prenyltransferase 1 (FcPT1) belongs to the UbiA superfamily, a prenyltransferase (PT) family of membrane-bound proteins possessing two aspartate-rich motifs that are conserved motifs crucial for the divalent cation-dependent prenylation [1].


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].


Usage and biology

FcPT1 is an umbelliferone dimethylallyltransferase (UDT), a PT that is specific for the first reaction step in the furanocoumarin (FC) biosynthetic pathway. It mediates a prenylation reaction in which umbelliferone is the acceptor of the prenyl group, and dimethylallylpyrophosphate (DMAPP) is the donor, producing DMS if the prenylation takes place at the 6th carbon position of umbelliferone, and osthenol if prenylation occurres at the 8th carbon position.


The team of Technion 2022 used this part in a construct (BBa_K4179003) 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 862
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1279
    Illegal SpeI site found at 862
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 862
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 862
    Illegal NgoMIV site found at 56
    Illegal NgoMIV site found at 621
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 137
    Illegal SapI site found at 933


This part includes the gene of FcPT1, 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 FcPT1 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 FcPT1 gene, due to the gene being cloned upstream to a P2A (BBa_K4179005)-mCherry (BBa_K106005) sequence. In this genetic system (BBa_K4179003) , the mCherry expression is tied directly to the start codon of FcPT1, 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 FcPT1’s expression.


References

1. Munakata R, Kitajima S, Nuttens A et al. Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants. New Phytologist 2020; 225: 2166–2182.

2. de Bruijn WJC, Levisson M, Beekwilder J, van Berkel WJH, Vincken J-P. Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories. 2020;

3. Bateman A, Martin MJ, Orchard S et al. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 2021; 49: D480–D489.