Deciphering Tryptophan Oxygenation: Key Modulators of 2‐Oxindole Formation in MarE

Abstract MarE, a heme‐dependent aromatic oxygenase with a histidyl axial ligation, catalyzes the monooxygenation of β‐methyl‐ l ‐tryptophan to form a 2‐oxindole scaffold central to maremycin biosynthesis. Although structurally similar to tryptophan 2,3‐dioxygenase (TDO), which initiates l ‐tryptophan catabolism via dioxygenation, MarE exhibits distinct reactivity modulated by ascorbate. While ascorbate has no effect on TDO, it promotes selective monooxygenation by MarE. In its absence, MarE favors dioxygenation and formation of pyrroloindoline products, revealing latent catalytic versatility. Active‐site loop sequences differ between the two enzymes, SLGGR in MarE versus GTGGS in TDO, prompting loop‐swapping experiments to probe structure‐function relationships. Substituting GTGGS in TDO to MarE‐like sequences (GTGGA or SLGGS) shifted reactivity toward monooxygenation and formation of C3‐hydroxylated, non‐oxindole products that underwent further cyclization into tricyclic structures. Conversely, replacing SLGGR in MarE with GTGGS resulted in enhanced C2,C3‐dioxygenation nearly 4‐fold. These results underscore the active‐site loop as a key determinant of oxidation outcome, alongside the modulatory role of ascorbate. By revealing the true catalytic identity of MarE and delineating the roles of small‐molecule effectors and loop architecture, this study advances mechanistic understanding and predictive capabilities within the oxygenase superfamily.