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  2. Supporting information: Chemical synthesis of trans 8-methyl-6-nonenoyl-CoA and functional expression unravel capsaicin synthase activity encoded by the Pun1 locus
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    Dataset: Supporting information: Chemical synthesis of trans 8-methyl-6-nonenoyl-CoA and functional expression unravel capsaicin synthase activity encoded by the Pun1 locus

    Alternate identifier:
    (InChIKey) YKPUWZUDDOIDPM-SOFGYWHQSA-N
    (InChI) InChI=1S/C18H27NO3/c1-14(2)8-6-4-5-7-9-18(21)19-13-15-10-11-16(20)17(12-15)22-3/h6,8,10-12,14,20H,4-5,7,9,13H2,1-3H3,(H,19,21)/b8-6+
    (CAS/RN) 404-86-4
    (InChIKey) PUDMGOSXPCMUJZ-UHFFFAOYSA-N
    (InChI) InChI=1S/C8H11NO2.ClH/c1-11-8-4-6(5-9)2-3-7(8)10;/h2-4,10H,5,9H2,1H3;1H
    (CAS/RN) 7149-10-2
    (InChIKey) JGNCYWQFXLYWMO-HSJNEKGZSA-N
    (InChI) InChI=1S/C31H52N7O17P3S/c1-19(2)9-7-5-6-8-10-22(40)59-14-13-33-21(39)11-12-34-29(43)26(42)31(3,4)16-52-58(49,50)55-57(47,48)51-15-20-25(54-56(44,45)46)24(41)30(53-20)38-18-37-23-27(32)35-17-36-28(23)38/h7,9,17-20,24-26,30,41-42H,5-6,8,10-16H2,1-4H3,(H,33,39)(H,34,43)(H,47,48)(H,49,50)(H2,32,35,36)(H2,44,45,46)/t20-,24-,25-,26+,30-/m1/s1
    Related identifier:
    (Is Supplement To) 10.3390/molecules27206878 - DOI
    (Documents) https://identifiers.org/pubchem.compound:1548943 - URL
    (Documents) https://identifiers.org/pubchem.compound:165576 - URL
    Creator/Author:
    Milde, Raika https://orcid.org/0000-0003-0630-8105 [Leibniz Institute of Plant Biochemistry, Dept. of Cell and Metabolic Biology, Weinberg 3, 06120 Halle (Saale), Germany]

    Schnabel, Arianne https://orcid.org/0000-0002-2172-2905 [Leibniz Institute of Plant Biochemistry, Dept. of Cell and Metabolic Biology, Weinberg 3, 06120 Halle (Saale), Germany]

    Ditfe, Toni https://orcid.org/0000-0001-6360-6673 [Leibniz Institute of Plant Biochemistry, Dept. of Bioorganic Chemistry, Weinberg 3, 06120 Halle (Saale), Germany]

    Hoehenwarter, Wolfgang https://orcid.org/0000-0002-7669-7524 [Leibniz Institute of Plant Biochemistry, Dept. of Biochemistry of Plant Interactions, Weinberg 3, 06120 Halle (Saale), Germany]

    Proksch, Carsten https://orcid.org/0000-0003-4948-8281 [Leibniz Institute of Plant Biochemistry, Dept. of Biochemistry of Plant Interactions, Weinberg 3, 06120 Halle (Saale), Germany]

    Westermann, Bernhard https://orcid.org/0000-0002-6228-5991 [Leibniz Institute of Plant Biochemistry, Dept. of Bioorganic Chemistry, Weinberg 3, 06120 Halle (Saale), Germany]

    Vogt, Thomas https://orcid.org/0000-0001-6155-3360 [Leibniz Institute of Plant Biochemistry, Dept. of Cell and Metabolic Biology, Weinberg 3, 06120 Halle (Saale), Germany]
    Contributors:
    -
    Title:
    Supporting information: Chemical synthesis of trans 8-methyl-6-nonenoyl-CoA and functional expression unravel capsaicin synthase activity encoded by the Pun1 locus
    Additional titles:
    -
    Description:
    (Abstract) Capsaicin, produced by diverse Capsicum species is among the world’s most popular spices and of considerable pharmaceutical relevance. Although the capsaicinoid biosynthetic pathway has been investigated for decades, several biosynthetic steps have remained partly hypothetical. Genetic evidence suggested that the decisive capsaicin synthase is encoded by the Pun1 locus. Yet, genetic evidence of the Pun1 locus was never corroborated by functionally active capsaicin synthase that presumably catalyzes amide bond formation between trans 8-methyl-6-nonenoyl-CoA derived from branched chain amino acid biosynthesis and vanilloylamine derived from the phenylpropanoid pathway. In this report, we demonstrate enzymatic activity of a recombinant capsaicin synthase encoded by Pun1, functionally expressed in E. coli, and provide information on substrate specificity and catalytic properties. Recombinant capsaicin synthase is specific for selected aliphatic CoA-esters and highly specific for vanilloylamine. Partly purified from E. coli, the recombinant active enzyme is a monomeric protein of 51 kDa that is independent of additional co-factors or associated proteins, as previously proposed. These data can now be used to design capsaicin synthase variants with different properties and alternative substrate preferences.
    Keywords:
    Capsaicin
    Capsicum
    Pun1
    acyltransferase
    BAHD
    CoA-ester
    enzyme activity
    substrate specificity
    Related information:
    -
    Language:
    -
    Publishers:
    Vogt, Thomas
    Production year:
    Subject areas:
    Biochemistry
    Resource type:
    (Dataset) chromatogram data (arw files), protein LC-MS/MS, images (tiff, jpeg), tabular data (csv, xlsx)
    Data source:
    (Instrument) Varian Mercury 400 NMR spectrometer (Agilent, Waldbronn, Germany)
    (Instrument) AB Sciex API 3200, Q-TRAP mass spectrometer (AB Sciex , Darmstadt, Germany)
    (Instrument) Waters e2695 chromatography work station equipped with a photodiode array detector (PDA) and a QDA-mass detector (Waters, Eschborn, Germany)
    (Instrument) Äkta Explorer FPLC (Cytiva, Freiburg, Germany) Instrument
    (Instrument) QExactive Plus mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany)
    (Organism) Capsicum
    (Organism) Escherichia coli
    Software used:
    Resource production
    Software:
    VnmrJ (Agilent) - 4.2 Revision A
    Alternative software:
    -
    Software used:
    Resource production
    Software:
    AB Sciex Analyst - 1.6.2
    Alternative software:
    -
    Software used:
    Resource processing
    Software:
    MestReNova - 14.0.1
    Alternative software:
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    Software used:
    Resource processing
    Software:
    ProteoWizard - 3.0.2
    Alternative software:
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    Software used:
    Resource production
    Software:
    Waters Empower (Waters) - 3
    Alternative software:
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    Software used:
    Resource production
    Software:
    Carl Zeiss Zen (Carl Zeiss AG) - 2012
    Alternative software:
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    Software used:
    Resource processing
    Software:
    Unicorn (Amersham Biosciences, now Cytiva ) - 5.0
    Alternative software:
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    Software used:
    Resource production
    Software:
    Mascot (Matrix Science) - v2.7.0
    Alternative software:
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    Software used:
    Resource production
    Software:
    Thermo Fisher Scientific Proteom Discoverer - v2.1
    Alternative software:
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    Software used:
    Resource processing
    Software:
    Microsoft Office - 2013
    Alternative software:
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    Software used:
    Resource processing
    Software:
    Systat SigmaPlot (Systat) - 14.0
    Alternative software:
    -
    Data processing:
    -
    Publication year:
    Rights holders:
    Leibniz Institute of Plant Biochemistry (IPB), Dept. Cell and Metabolic Biology and Dept. Bioorganic Chemistry, Weinberg 3, 06120 Halle (Saale), Germany.
    Funding:
    -
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    Status:
    Published
    Uploaded by:
    1670fa88949cc897f41b44d58d08c11a
    Created on:
    Archiving date:
    2022-10-19
    Archive size:
    4.0 GB
    Archive creator:
    1670fa88949cc897f41b44d58d08c11a
    Archive checksum:
    16327379e5033c24cc9cb2ccbfd4a5f1 (MD5)
    Embargo end date:
    -