Difference between revisions of "Part:BBa K1131006"
 
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<partinfo>BBa_K1131006 short</partinfo>
 
<partinfo>BBa_K1131006 short</partinfo>
  
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This oleandomycin glucosyltransferase was found to glucosylate indoxyl to produce indoxyl-B-glucoside, or indican. To the best of our knoweldge, this is the first recombinant enzyme used to produce indican. Kaempferol is another substrate of this enzyme. It is known to highly promiscuous in substrate specificity and glucosylates a variety of phenolics and anthrocyanins. The sequence shown here corresponds to that of UniProt ID Q3HTL6, and encodes the ORF only. This is the characterized and slightly altered version of [https://parts.igem.org/Part:BBa_K1131004 BBa1131004]. The last 15 amino acids have been removed from this version increasing its solubility greatly. A mutated version known to have even broader activity was [https://parts.igem.org/Part:BBa_K1131007 added to registry] as well.
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This oleandomycin glucosyltransferase was found to glucosylate indoxyl to produce indoxyl-B-glucoside, or indican. To the best of our knoweldge, this is the first recombinant enzyme used to produce indican. Kaempferol is another substrate of this enzyme. It is known to highly promiscuous in substrate specificity and glucosylates a variety of phenolics and anthrocyanins. The sequence shown here corresponds to that of UniProt ID Q3HTL6, and encodes the ORF only. This is the characterized and slightly altered version of [https://parts.igem.org/Part:BBa_K1131004 BBa_K1131004]. The last 15 amino acids have been removed from this version increasing its solubility greatly. A mutated version known to have even broader activity was [https://parts.igem.org/Part:BBa_K1131007 added to the Registry] as well.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1131006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1131006 SequenceAndFeatures</partinfo>
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[[File:OleD-K-UDPG-bleck-short.png|500px]]<br>
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Absorbance data obtained from HPLC at 270nm. Reaction containing Kaempferol, OleD, and donor molecule UDP-glucose. Peaks for kaempferol and kaempferol glucoside are observed demonstrating the successful purification of active OleD.
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[[File:HPLC_Indican_Black_1.png|500px]]<br>
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Absorbance data obtained from HPLC at 270nm. Reaction containing indoxyl, OleD, and donor molecule UDP-glucose peaks for indican and sodium complexed indican are observed along with the precursor indoxyl.<br>
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[[File:T4_IPO4_UDPG_Black_1.png|500px]]<br>
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Mass spectrometry data for the elution fraction at a retention time of 9.51 (Indican peak in HPLC data shown in previous figure). Peaks matching the mass of the precursor indoxyl (134), indican (296), and sodium complexed indican (318) were found. All unlabeled peaks did not correspond to expected ions.<br>
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1131006 parameters</partinfo>
 
<partinfo>BBa_K1131006 parameters</partinfo>
 
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Latest revision as of 04:57, 1 November 2013

oleD glucosyltransferase; S. antibiotics (characterized)

This oleandomycin glucosyltransferase was found to glucosylate indoxyl to produce indoxyl-B-glucoside, or indican. To the best of our knoweldge, this is the first recombinant enzyme used to produce indican. Kaempferol is another substrate of this enzyme. It is known to highly promiscuous in substrate specificity and glucosylates a variety of phenolics and anthrocyanins. The sequence shown here corresponds to that of UniProt ID Q3HTL6, and encodes the ORF only. This is the characterized and slightly altered version of BBa_K1131004. The last 15 amino acids have been removed from this version increasing its solubility greatly. A mutated version known to have even broader activity was added to the Registry as well.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 688
    Illegal NgoMIV site found at 928
    Illegal NgoMIV site found at 1118
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 387
    Illegal BsaI.rc site found at 653
    Illegal BsaI.rc site found at 1078

OleD-K-UDPG-bleck-short.png
Absorbance data obtained from HPLC at 270nm. Reaction containing Kaempferol, OleD, and donor molecule UDP-glucose. Peaks for kaempferol and kaempferol glucoside are observed demonstrating the successful purification of active OleD. HPLC Indican Black 1.png
Absorbance data obtained from HPLC at 270nm. Reaction containing indoxyl, OleD, and donor molecule UDP-glucose peaks for indican and sodium complexed indican are observed along with the precursor indoxyl.
T4 IPO4 UDPG Black 1.png
Mass spectrometry data for the elution fraction at a retention time of 9.51 (Indican peak in HPLC data shown in previous figure). Peaks matching the mass of the precursor indoxyl (134), indican (296), and sodium complexed indican (318) were found. All unlabeled peaks did not correspond to expected ions.