Journal Article


The formation of a camalexin-biosynthetic metabolon

Abstract

Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway by formation of an enzyme complex. To identify such protein interactions, two independent untargeted co49 immunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B1 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were shown to co-purify. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, interaction of CYP71A13 and Arabidopsis P450 Reductase 1 (ATR1) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating allosteric interaction. Camalexin biosynthesis involves glutathionylation of an intermediary indole-3-cyanohydrin, synthesized by CYP71A12 and especially CYP71A13. It was demonstrated by FRET-FLIM and co-IP, that the glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism.

Attached files

Authors

Mucha, Stefanie
Heinzlmeir, Stephanie
Kriechbaumer, Verena
Strickland, Benjamin
Kirchhelle, Charlotte
Choudhary, Manisha
Kowalski, Natalie
Eichmann, Ruth
Hückelhoven, Ralph
Grill, Erwin
Küster, Bernhard
Glawischnig, Erich

Oxford Brookes departments

Department of Biological and Medical Sciences

Dates

Year of publication: 2019
Date of RADAR deposit: 2019-09-03


Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License


Related resources

This RADAR resource is the Accepted Manuscript of The formation of a camalexin-biosynthetic metabolon
This RADAR resource is the Accepted Manuscript of The formation of a camalexin-biosynthetic metabolon

Details

  • Owner: Joseph Ripp
  • Collection: Outputs
  • Version: 1 (show all)
  • Status: Live