Difference between revisions of "Part:BBa K4179000"
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Prenylates umbelliferone in the presence of dimethylallyl diphosphate (DMAPP) at the 6 position to yield demethylsuberosin (DMS) as the main product, together with a minor amount of osthenol corresponding to 8-prenylated umbelliferone.  
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This sequence codes for a prenyl transferase. An enzyme which prenylates umbelliferone in the presence of dimethylallyl diphosphate (DMAPP) at the 6th carbon position to produce demethylsuberosin (DMS) as the main product, in addition to a minor amount of osthenol corresponding to umbelliferone prenylated at the 8th carbon position.
  
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==Prenylation==
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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.  
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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.
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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.  
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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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==Characterization==
 
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PcPT is a transmembrane proteins with 8 predicted transmembrane helices. The gene was first isolated by designing degenerate primers based on conserved amino acids in other already identified aromatic PTs. Using these primers, a PCR-based strategy was used to clone a PT cDNA  from  parsley (Petroselinum crispum)  leaves,  which  was  named PcPT. PcPT gene expression was found throughout the parsley plant, and was increased by UV irradiation. The subcellular localization of PcPT was demonstrated to be plastids.
 
  
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===Usage and biology===
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The gene of PcPT, Petroselinum crispum prenyl transferase, was first isolated from the genome of parsley leaves by Karamat et al and codon optimized for E. coli. It is a transmembrane protein with 8 predicted transmembrane helices. Its subcellular localization was found to be plastids.
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Functional characterization of PcPT done by Karamat et al showed that PcPT has a strong substrate specificity for DMAPP as a prenyl donor, with a dominant prenylation activity of umbelliferone at the 6th position, yielding DMS as the major product.
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The enzyme contains a cleavage site for a signal peptide sequence (48 aa) at the N-terminus.
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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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Functional characterization of PcPT in a transient expression system with Nicotiana benthamiana showed that PcPT shows prenylation activity for umbelliferone with strong substrate specificity for dimethylallyl diphosphate (DMAPP) as a prenyl donor, dominant prenylation activity on umbelliferone at C6 leading to DMS, and minor activity at C8 of umbelliferone leading to osthenol.
 
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PcPT contains a cleavage site for a signal peptide sequence (48 aa) at the N-terminus. Using GFP-fusion based experiments, the results strongly suggest that PcPT functions in plastids, where it utilized DMAPP as a prenyl donor.
 
  
  
  
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===Usage and Biology===
 
  
 
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<!-- Uncomment this to enable Functional Parameter display
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This part includes the gene of PcPT, 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 PcPT 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_K4179000 parameters</partinfo>
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A stop codon is not present at the end of the PcPT 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 PcPT, 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 PcPT’s expression.
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Revision as of 14:32, 5 October 2022


Petroselinum crispum prenyl transferase (PcPT)

Structure of PcPT enzyme


This sequence codes for a prenyl transferase. An enzyme which prenylates umbelliferone in the presence of dimethylallyl diphosphate (DMAPP) at the 6th carbon position to produce demethylsuberosin (DMS) as the main product, in addition to a minor amount of osthenol corresponding to umbelliferone prenylated at the 8th carbon position.

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.


Usage and biology

The gene of PcPT, Petroselinum crispum prenyl transferase, was first isolated from the genome of parsley leaves by Karamat et al and codon optimized for E. coli. It is a transmembrane protein with 8 predicted transmembrane helices. Its subcellular localization was found to be plastids. Functional characterization of PcPT done by Karamat et al showed that PcPT has a strong substrate specificity for DMAPP as a prenyl donor, with a dominant prenylation activity of umbelliferone at the 6th position, yielding DMS as the major product. The enzyme contains a cleavage site for a signal peptide sequence (48 aa) at the N-terminus.

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 559
    Illegal EcoRI site found at 922
    Illegal PstI site found at 82
    Illegal PstI site found at 445
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 559
    Illegal EcoRI site found at 922
    Illegal NheI site found at 1207
    Illegal PstI site found at 82
    Illegal PstI site found at 445
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 559
    Illegal EcoRI site found at 922
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 559
    Illegal EcoRI site found at 922
    Illegal PstI site found at 82
    Illegal PstI site found at 445
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 127
    Illegal EcoRI site found at 559
    Illegal EcoRI site found at 922
    Illegal PstI site found at 82
    Illegal PstI site found at 445
  • 1000
    COMPATIBLE WITH RFC[1000]


This part includes the gene of PcPT, 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 PcPT 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 PcPT gene, due to the gene being cloned upstream to a P2A (BBa_K4179005)-mCherry [ https://parts.igem.org/wiki/index.php?title=Part:BBa_K106005 (BBa_K106005)] sequence. In this genetic system (BBa_K4179003) , the mCherry expression is tied directly to the start codon of PcPT, 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 PcPT’s expression.