Abstract
Thaxtomins, a family of phytotoxins produced by Streptomyces spp., can cause dramatic plant cell hypertrophy and seedling stunting. Thaxtomin A is the dominant form from Streptomyces scabies and has demonstrated herbicidal action. TxtE, a cytochrome P450 enzyme from Streptomyces scabies 87.22, catalyzes direct nitration of the indolyl moiety of L-tryptophan to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH. The crystal structure of TxtE was determined at 2.1 Å resolution and described in this work. A clearly defined substrate access channel is observed and can be classified as channel 2a, which is common in bacteria cytochrome P450 enzymes. A continuous hydrogen bond chain from the active site to the external solvent is observed. Compared with other cytochrome P450 enzymes, TxtE shows a unique proton transfer pathway which crosses the helix I distortion. Polar contacts of Arg59, Tyr89, Asn293, Thr296, and Glu394 with L-tryptophan are seen using molecular docking analysis, which are potentially important for substrate recognition and binding. After mutating Arg59, Asn293, Thr296 or Glu394 to leucine, the substrate binding ability of TxtE was lost or decreased significantly. Based on the docking and mutation results, a possible mechanism for substrate recognition and binding is proposed.
Highlights
Cytochromes P450 superfamily (CYP) is a large and diverse group of enzymes containing a heme cofactor which participate in a variety of physiological roles including hormone and secondary metabolite biosynthesis, as well as xenobiotic metabolism[1,2]
Six proteins were identified in the Thaxtomin A biosynthesis pathway: TxtA and TxtB are nonribosomal peptide synthases[6,7]; TxtC[8] and TxtE[9] are CYPs; TxtD is a nitric oxide synthase that produces nitric oxide from L-arginine[10]; TxtR is a cellobiose-responsive pathway specific activator[11]
TxtE catalyzes direct nitration of the indolyl moiety of L-tryptophan (L-Trp) to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH (Figure 1), which is the key step of Thaxtomin A biosynthesis
Summary
Cytochromes P450 superfamily (CYP) is a large and diverse group of enzymes containing a heme cofactor which participate in a variety of physiological roles including hormone and secondary metabolite biosynthesis, as well as xenobiotic metabolism[1,2]. Most of CYPs catalyze the insertion of an oxygen atom from dioxygen into the carbon-hydrogen bonds of organic compounds, while the other oxygen atom reacts with protons to form water. This reaction is referred to as the monooxygenase reaction. TxtE catalyzes direct nitration of the indolyl moiety of L-tryptophan (L-Trp) to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH (Figure 1), which is the key step of Thaxtomin A biosynthesis. TxtE only catalyzes a nitration reaction at the 4-position of indolyl moiety. Combined with the catalytic mechanism of CYPs, it was proposed that TxtE may activate dioxygen and nitric oxide to form a peroxynitrite[9]
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