Thaxtomins, a family of phytotoxins produced by Streptomyces spp., can cause plant cell necrosis at nanomolar concentrations. In the thaxtomin biosynthesis pathway, six proteins were identified. TxtE, a cytochrome P450 enzyme from S. scabies 87.22, catalyzes direct and regiospecific nitration of the indolyl moiety of L-tryptophan to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH, which is the key step of thaxtomin biosynthesis.
TxtE is the first reported enzyme that catalyzes a direct nitration reaction in the biosynthetic pathway and it has the potential to be developed for industrial applications. Here we report the crystal structure of TxtE determined at 2.1Å to understand the possible mechanism of its regiospecific nitration of the substrates. Based on the structure, 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 surface of TxtE is observed. Compared with other cytochrome P450 enzymes, TxtE shows a unique proton transfer pathway, which crosses the helix I distortion. Using molecular docking analysis, we find the polar contacts of Arg59, Tyr89, Asn293, Thr296, and Glu394 with L-tryptophan are important for substrate recognition and binding. After mutating Arg59, Asn293, Thr296 or Glu394 to leucine, the substrate binding ability of TxtE is shown to disappear or decreased significantly. Combined with the structure and mutagenesis experiment results, a possible mechanism for substrate recognition and binding is proposed.