Tryptophan indole-lyase (TIL) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible β-elimination of L-tryptophan into indole, pyruvate, and ammonia. This enzyme has significant potential for the synthesis of L-tryptophan and its derivatives, which are valuable in pharmaceutical, agricultural, and fine chemical applications. Here, a metagenome derived from chicken manure and straw compost was used to mine for a TIL. We combined molecular cloning and computational modeling to identify and characterize a novel TIL, aiming to develop a technology for the production of L-tryptophan and its derivatives. The TIL gene was cloned into the pET-24a(+) vector using Gibson assembly and expressed in E. coli BL21 Star cells. We demonstrated the TIL capability for both tryptophan synthesis and degradation, with specific activities of 0.022 U/mg and 0.69 U/mg, respectively. Using AlphaFold 2, a 3D model of TIL was generated and evaluated. Further, L-tryptophan and a panel of substituted analogs were analyzed by molecular docking. Among tested analogs 7-Aza-L-tryptophan showed the highest affinity (-8.37 kcal/mol) towards the enzyme. This enzyme-ligand complex and ligand-free enzyme were subjected to 100 ns MD simulation and compared. Structural stability, flexibility, compactness, and solvent exposure were evaluated for both systems, revealing stability throughout the simulation. Protein-ligand hydrogen bond formation showed stable interactions across the simulation. MM-PBSA analysis demonstrated an energetically favorable interaction (-37.35 kcal/mol); additionally, individual residue contributions were analyzed. Together, in vitro and in silico analyses confirm TIL functionality and catalytic potential, supporting further biochemical characterization and engineering for sustainable biocatalysis.

Co-authors: Artur Hambardzumyan, Tigran Soghomonyan, Marina Paronyan, Anichka Hovsepyan, Vladimir Vukic, Haykanush Koloyan