Reported by Yan-Yan Zhang from Yangzhou University of China

Abstract: Herein, we report an engineered enzyme that can monooxygenate unprotected tryptophan into the corresponding3a-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.

In conclusion, by evaluating the stepwise oxygen-insertion mechanism of tryptophan dioxygenases and taking advantage of directed evolution, we interrupted the dual oxygeninsertion process and transformed the xcTDO tryptophan dioxygenase into a monooxygenase. Our engineered xcTDO exhibits excellent TON and high diastereoselectivity, and enables the scalable synthesis of HPICs from Trps in a single step. To our knowledge, this is the first enzyme that can catalyze the oxidative cyclization of unprotected Trps in a highly diastereoselective fashion, a process that has not been synthetically realized before. Residue F51 was identified as being critical in determining the HPIC/NFK product distribution and enzyme activity. Furthermore, both our reaction and purification processes require no organicsolvent, making this an eco-friendly method for synthesizing HPICs for further applications.

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