Directed Evolution of a Tryptophan 2,3‐Dioxygenase for the Diastereoselective Monooxygenation of Tryptophans

Abstract Herein, we report an engineered enzyme that can monooxygenate unprotected tryptophan into the corresponding 3a‐hydroxyhexahydropyrrolo[2,3‐b]indole‐2‐carboxylic acid (HPIC) in a single, scalable step with excellent turnover number and diastereoselectivity. Taking advantage of directed evolution, we analyzed the stepwise oxygen‐insertion mechanism of tryptophan 2,3‐dioxygenases, and transformed tryptophan 2,3‐dioxygenase from Xanthomonas campestris into a monooxygenase for oxidative cyclization of tryptophans. It was revealed that residue F51 is vital in determining the product ratio of HPIC to N ′‐formylkynurenine. Our reactions and purification procedures use no organic solvents, resulting in an eco‐friendly method to prepare HPICs for further applications.