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

DOI

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.

Varian Mercury 400 NMR spectrometer (Agilent, Waldbronn, Germany)

AB Sciex API 3200, Q-TRAP mass spectrometer (AB Sciex , Darmstadt, Germany)

Waters e2695 chromatography work station equipped with a photodiode array detector (PDA) and a QDA-mass detector (Waters, Eschborn, Germany)

Äkta Explorer FPLC (Cytiva, Freiburg, Germany) Instrument

QExactive Plus mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany)

Capsicum

Escherichia coli

Identifier
DOI https://doi.org/10.22000/799
Related Identifier https://doi.org/10.3390/molecules27206878
Related Identifier https://identifiers.org/pubchem.compound:1548943
Related Identifier https://identifiers.org/pubchem.compound:165576
Metadata Access https://www.radar-service.eu/oai/OAIHandler?verb=GetRecord&metadataPrefix=datacite&identifier=10.22000/799
Provenance
Creator Milde, Raika ORCID logo; Schnabel, Arianne ORCID logo; Ditfe, Toni ORCID logo; Hoehenwarter, Wolfgang ORCID logo; Proksch, Carsten ORCID logo; Westermann, Bernhard ORCID logo; Vogt, Thomas ORCID logo
Publisher Vogt, Thomas
Contributor RADAR
Publication Year 2022
Rights Open Access; Creative Commons Attribution 4.0 International; info:eu-repo/semantics/openAccess; https://creativecommons.org/licenses/by/4.0/legalcode
OpenAccess true
Representation
Resource Type chromatogram data (arw files), protein LC-MS/MS, images (tiff, jpeg), tabular data (csv, xlsx); Dataset
Format application/x-tar
Discipline Basic Biological and Medical Research; Biochemistry; Biology; Life Sciences