Cytochrome P450–catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis

Abstract

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N'-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O(2).

Figure 1

Thaxtomin biosynthesis. a: Organization of the thaxtomin biosynthetic gene cluster in S. turgidiscabies. Biosynthetic genes are in black; txtR, which encodes a cellobiose-responsive pathway specific activator, is in dark gray; two genes encoding putative transposases are in light gray. The txtAB genes encode nonribosomal peptide synthetases. The txtC and txtE genes encode CYPs. The txtD gene encodes a NOS that produces NO from L-arginine. b: Proposed pathway for thaxtomin A biosynthesis.

Figure 2

Characterization of purified recombinant TxtE. a: Extracted ion chromatograms at m/z = 204 and 250 (right) from LC-MS analyses of the enzymatic nitration reaction (left). The bottom chromatogram is from the enzymatic reaction and the top chromatogram is from a negative control containing boiled TxtE. b: High resolution mass spectra of L-4-nitrotryptophan obtained from incubation of L-tryptophan with TxtE, DEANO, ferredoxin, ferredoxin reductase and NADPH in air (top spectrum), and in an ¹⁸O₂ atmosphere (bottom spectrum). The m/z = 250.0818 ion corresponds to [M+H]⁺ for L-4-nitrotryptophan. An ion corresponding to [M+H]⁺ for L-4-nitrotryptophan containing a single ¹⁸O label (m/z calculated for C₁₁H₁₂N₃O ⁺₄: 252.0865; found: 252.0853) predominates (>90%) in the bottom spectrum c: Comparison of the proposed mechanism of TxtE-catalyzed nitration and the mechanism of CYP-catalyzed hydroxylation. Intermediates that differ in CYP-catalyzed hydroxylation and nitration are highlighted in red and blue, respectively. Common intermediates are black. The atoms of O₂ are filled to illustrate their fate in the reactions.