The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network ofArabidopsis thaliana   

Abstract

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, g-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys (IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.

Assigning Gene Function in Biosynthetic Pathways: Camalexin and Beyond

“…Only proper identification of the carboxypeptidase will allow complete elucidation of the camalexin biosynthesis puzzle. Bö ttcher et al (2009) demonstrated the presence of g-Glu-Cys (IAN)-Gly, g-Glu-Cys(IAN), Cys(IAN), and dihydrocamalexic acid in AgNO 3 -induced Arabidopsis plants. This set of compounds corresponds to all possible intermediates in the pathway from IAN to camalexin except for Cys(IAN)-Gly.…”

“…Subsequently, CYP71A13 converts the aldoxime to IAN (Nafisi et al, 2007), which, after an unknown activation step, is conjugated to the tripeptide glutathione (g-Glu-Cys-Gly) by glutathione Stransferases, including GSTF6 (Bö ttcher et al, 2009;Su et al, 2011). The glutathione conjugate g-Glu-Cys(IAN)-Gly is then hydrolyzed by two consecutive peptidase reactions (discussed in detail below) to Cys(IAN), which is finally converted to camalexin by a two-step reaction catalyzed by CYP71B15 (PAD3) (Zhou et al, 1999;Schuhegger et al, 2006;Bö ttcher et al, 2009) (Figure 1). …”

“…All rights reserved. camalexin, can be further modified, for example, by hydroxylation or glucosylation (Bö ttcher et al, 2009). To provide experimental evidence for the involvement of GGTs, Su et al (2011) analyzed camalexin levels (end-product levels) in media of liquid-cultured single T-DNA knockout mutants of ggt1 and ggt2 and found a decrease of 40 to 60% in both mutants.…”

“…Rosette leaves have been shown to express the camalexin biosynthetic machinery after treatment with AgNO 3 or infection with B. cinerea and are accordingly the most used tissue for camalexin studies (Glawischnig et al, 2004;Schuhegger et al, 2006;Bö ttcher et al, 2009), though other tissues, such as roots, are also known to accumulate camalexin upon induction (Bednarek et al, 2005). To complement the existing expression data for both GGP1 and the GGTs, we investigated the expression of GGT1, GGT2, and GGP1 transcripts in rosette leaves upon camalexin induction using an in silico approach.…”

“…The pathway leading to this model phytoalexin has been almost fully elucidated, and recent focus has been on the biosynthetic origin of the thiazole ring. Several publications have shown that glutathione, and not Cys, is the direct source of the heterocycle and have demonstrated that both glutathione and Cys conjugates of indole-3-acetonitrile (IAN) are intermediates in the pathway (Bö ttcher et al, 2009;Geu-Flores et al, 2011;Su et al, 2011). However, two recent studies published in this journal arrived at different conclusions regarding the conversion of the glutathione conjugate (g-Glu-Cys[IAN]-Gly) to the Cys conjugate (Cys [IAN]), particularly regarding the family of enzymes cleaving off the g-Glu residue.…”