CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis

Abstract

Camalexin represents the main phytoalexin in Arabidopsis (Arabidopsis thaliana). The camalexin-deficient phytoalexin deficient 3 (pad3) mutant has been widely used to assess the biological role of camalexin, although the exact substrate of the cytochrome P450 enzyme 71B15 encoded by PAD3 remained elusive. 2-(Indol-3-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid (dihydrocamalexic acid) was identified as likely intermediate in camalexin biosynthesis downstream of indole-3-acetaldoxime, as it accumulated in leaves of silver nitrate-induced pad3 mutant plants and it complemented the camalexin-deficient phenotype of a cyp79b2/cyp79b3 double-knockout mutant. Recombinant CYP71B15 heterologously expressed in yeast catalyzed the conversion of dihydrocamalexic acid to camalexin with preference of the (S)-enantiomer. Arabidopsis microsomes isolated from leaves of CYP71B15-overexpressing and induced wild-type plants were capable of the same reaction but not microsomes from induced leaves of pad3 mutants. In conclusion, CYP71B15 catalyzes the final step in camalexin biosynthesis.

Figure 1.

The camalexin biosynthetic pathway. Cys-R, Cys or Cys derivative.

Figure 2.

LC-MS analysis of dihydrocamalexic acid in methanol extracts of rosette leaves of pad3 and wild-type plants 18 h after silver nitrate spraying. A and B, Extracted ion chromatogram (m/z = 247) of wild type (A) and pad3 mutants (B) leaf extract is shown. C, Dihydrocamalexic acid standard. One of three independent experiments with comparable results is presented.

Figure 3.

Analysis of CYP71B15 activity. A, HPLC profile with fluorescence detection after enzymatic conversion of (S)-dihydrocamalexic acid to camalexin by microsomes from yeast expressing CYP71B15 in the presence and absence of NADPH, or from yeast vector control (with NADPH). The product identity was confirmed by UV spectroscopy (B) and electron ionization MS spectrometry (C).

Figure 4.

Kinetic properties of CYP71B15. The conversion of the (S)- and (R)-enantiomer of dihydrocamalexic acid to camalexin by microsomes of yeast expressing CYP71B15 (A) and Arabidopsis (B) was determined for different substrate concentrations. Substrate turnover (pmol mg⁻¹ min⁻¹) is plotted against substrate concentration (μm). Squares, (S)-enantiomer; circles, (R)-enantiomer.

Figure 5.

Camalexin formation from (S)-dihydrocamalexic acid by Arabidopsis microsomes. Camalexin formation was compared from microsomes of wild type, 35S∷CYP71B15, and pad3 mutant. Left, Fluorescence chromatogram (using microsomes of the named genotypes with and without 16 h silver nitrate induction). For all preparations, NADPH-independent background was observed. Right, NADPH-dependent enzymatic activity of the six preparations (designated left, each average of three tests).

Figure 6.

Analysis of CYP71B15 expression in CYP71B15p∷GUS plants. A to E, GUS staining after challenging leaves with droplets of A. alternata spore suspension (A), P. syringae DC3000/Rps4 cell suspension (C), and 5 mm silver nitrate solution (E). Localized CYP71B15 induction was observed. Corresponding control treatments with buffer are shown in B and D, respectively, indicating that tween-containing buffer induces a minor CYP71B15 induction. Scale bars = 2 mm. F to H, GUS staining in roots, untreated (F and G) or 16 h after challenging with 5 mm silver nitrate (H). Scale bars = 0.2 mm.

Figure 7.

Proposed mechanism for CYP71B15. The pentavalent oxoiron in the reaction center initially abstracts a hydrid ion from C-5 of the thiazole ring. The formed intermediate liberates carbon dioxide in a fast spontaneous process forming a C-4/C-5 double bond.