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.

中文翻译：

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针对色氨酸的非对映选择性单加氧作用的色氨酸2，3-双加氧酶的定向进化。

在这里，我们报告了一种工程酶，它可以在单一的，可扩展的步骤中将未保护的色氨酸单加氧成相应的3a-羟基六氢吡咯并[2,3-b]吲哚-2-羧酸（HPIC），具有出色的周转数和非对映选择性。利用定向进化的优势，我们分析了色氨酸2，3-双加氧酶的逐步氧插入机理，并将来自油菜黄单胞菌的色氨酸2，3-双加氧酶转化为用于色氨酸的氧化环化的单加氧酶。揭示了残基F51对于确定HPIC与N'-甲酰基kynurenine的产物比率至关重要。我们的反应和纯化程序不使用有机溶剂，因此采用了环保的方法来制备HPIC，以供进一步应用。