Structural Insights into the Mechanism for Recognizing Substrate of the Cytochrome P450 Enzyme TxtE

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.

Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE

“…used AutoDock to propose three L-tryptophan binding poses. [13] The binding orientation observed in our study closely resembles their first two proposals. As shown in Figure 2b, the tryptophan (which is bound to approximately 80% occupancy) binds with its indole side chain facing into the distal face of the heme, displacing the water that ligates the heme (this water is still visible in the tryptophan-bound structure, refined at 20% occupancy).…”

“…Residues Met88 and Phe395 define the hydrophobic cleft for the substrate’s indole moiety. The C-4 position of the indole ring, the site of nitration, faces away from the heme and toward the hydroxyl of Tyr89 and the solvent channel that was identified previously [13] and confirmed in our structures. In the substrate-free structure, three ordered waters define this solvent channel as reported, [13] whereas in the tryptophan-bound structure a tube of indistinct density is visible that could not be modeled as waters.…”

“…[13] Here, we were able to crystallize the enzyme in a substrate-free state using a mixture of lithium sulfate and ammonium sulfate as a precipitant. Diffraction data were collected both with and without the addition of L-tryptophan.…”

“…The structures described here are in agreement with the previously reported structure (backbone RMSD 0.237 Å and 0.219 Å, respectively, to chain A of 4L36), while providing a higher resolution picture of the protein active site in the absence and presence of the native substrate. A detailed discussion of the fold and active site structure can be found in the report of Yu et al ; [13] here we will focus on how the enzyme interacts with its substrate, which can only be seen in the current structures.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…The second P450, TxtE, 274,301 performs an even more intriguing transformation: the regiospecic 4-nitration of the indolyl moiety of L-tryptophan. 274 Subsequent studies have also shown that TxtE has tolerance for certain alternate substrates.…”