Difference between revisions of "Part:BBa K4179001"

 
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<partinfo>BBa_K4179001 short</partinfo>
 
<partinfo>BBa_K4179001 short</partinfo>
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[[Image:PsPT1 image.png|thumb|400px|'''Structure of PsPT1 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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P. sativa prenyltransferase 1 (PsPT1) was discovered and characterized as a plastidic prenyltransferase, that synthesizes demethylsuberosin (DMS), a linear furanocoumarin (FC) intermediate, and osthenol an angular FC intermediate, respectively, in parsnip [1].
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===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 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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P. sativa prenyltransferase 1 (PsPT1) was discovered and characterized as a plastidic prenyltransferase, with 6 predicted transmembrane helices, that synthesize demethylsuberosin (DMS), a linear furanocoumarin (FC) intermediate, and osthenol an angular FC intermediate, respectively, in parsnip.
 
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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 prenyltransferases (PTs) that transfer isoprenoid moieties from pyrophosphate donor substrates to aromatic acceptor substrates.
 
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PsPT1 is an umbelliferone dimethylallyltransferase (UDT), a prenyltransferase that is specific for the first reaction step in the 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 of the prenylation took place at the C6 position of umbelliferone, and osthenol if prenylation occurred in the C8 position.
 
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Crude leaf microsomes were prepared by Munakata et al., to investigate angular FC synthesis in parsnips. Incubation of the microsomes with umbelliferone, DMAPP, and the cofactors required for the reaction generated DMS and, to a lesser extent, osthenol, thus indicating that the UDTs in parsnip favor the formation of DMS over osthenol.
 
  
  
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===Usage and Biology===
 
===Usage and Biology===
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PsPT1 is an umbelliferone dimethylallyltransferase (UDT), a prenyltransferase that is specific for the first reaction step in the FC biosynthetic pathway. It catalyzes a prenylation reaction with umbelliferone as the acceptor molecule and dimethylallylpyrophosphate (DMAPP) as the donor of the prenyl group, producing DMS if the prenylation took place at the 6th carbon position of umbelliferone, and osthenol if prenylation occurred at the 8th carbon position.
  
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<span class='h3bb'>Sequence and Features</span>
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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_K4179001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4179001 SequenceAndFeatures</partinfo>
  
  
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This part includes the gene of PsPT1, 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 PsPT1 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_K4179001 parameters</partinfo>
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A stop codon is not present at the end of the PsPT1 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/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 PsPT1, 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 PsPT1’s expression.
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==References==
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1. Munakata R, Olry A, Karamat F et al. Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis. New Phytologist 2016; 211: 332–344.
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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 15:20, 5 October 2022


Pastinaca sativa prenyl transferase 1 (PsPT1)

Structure of PsPT1 enzyme [3]
Prenylation of umbelliferone using DMAPP as a prenyl donor


P. sativa prenyltransferase 1 (PsPT1) was discovered and characterized as a plastidic prenyltransferase, that synthesizes demethylsuberosin (DMS), a linear furanocoumarin (FC) intermediate, and osthenol an angular FC intermediate, respectively, in parsnip [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

PsPT1 is an umbelliferone dimethylallyltransferase (UDT), a prenyltransferase that is specific for the first reaction step in the FC biosynthetic pathway. It catalyzes a prenylation reaction with umbelliferone as the acceptor molecule and dimethylallylpyrophosphate (DMAPP) as the donor of the prenyl group, producing DMS if the prenylation took place at the 6th carbon position of umbelliferone, and osthenol if prenylation occurred 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 EcoRI site found at 127
    Illegal EcoRI site found at 562
    Illegal PstI site found at 55
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 562
    Illegal NheI site found at 1210
    Illegal PstI site found at 55
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 562
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 562
    Illegal PstI site found at 55
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 562
    Illegal PstI site found at 55
  • 1000
    COMPATIBLE WITH RFC[1000]


This part includes the gene of PsPT1, 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 PsPT1 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 PsPT1 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 PsPT1, 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 PsPT1’s expression.


References

1. Munakata R, Olry A, Karamat F et al. Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis. New Phytologist 2016; 211: 332–344.

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.