Structural Snapshots of Tryptophan Indole-Lyase Reveal Insights into the Catalytic Mechanism.

Tryptophan indole lyase (TIL; [E.C. 4.1.99.1]) is a bacterial pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes reversible β-elimination of indole from L-tryptophan. The mechanism of elimination of indole from L-tryptophan starts with the formation of an external aldimine of the substrate and PLP, followed by deprotonation of the α-CH of the substrate, forming a resonance-stabilized quinonoid intermediate. Proton transfer to C3 of the indole ring and carbon-carbon bond cleavage of the quinonoid intermediate provide indole and aminoacrylate bound to PLP, which then releases indole, followed by iminopyruvate. We have now determined the X-ray crystal structures of TIL complexes with (3)-dioxindolyl-l-alanine, an inhibitor, and with substrates L-tryptophan, 7-aza-L-tryptophan, and -ethyl-l-cysteine (SEC) in the presence of benzimidazole (BZI), an isostere of the product indole. These structures show a mixture of -diamine, external aldimine, quinonoid, and aminoacrylate intermediates, in both open and closed active site conformations. In the closed conformations of L-tryptophan, (3)-dioxindolyl-l-alanine, and 7-aza-L-tryptophan complexes, hydrogen bonds form between Asp-133 with N1 of the ligand heterocyclic ring and NE2 of His-458 in the small domain of TIL. This hydrogen bond also forms in the BZI complex with the aminoacrylate intermediates formed from both L-tryptophan and SEC. The closed quinonoid complex of 7-aza-L-tryptophan shows that the azaindole ring in the closed conformation is bent out of plane of the Cβ-C3 bond by about 40°, putting it in a geometry that leads toward the transition-state geometry. Thus, both conformational dynamics and substrate activation play critical roles in the reaction mechanism of the TIL.