# Structural Basis of Regioselective Bromination of Tricyclic Tryptoline by the Tryptophan Halogenase Thal

## TL;DR

This study shows how a halogenase enzyme can efficiently and selectively brominate a non-native compound, tryptoline, and explains the structural basis for this selectivity.

## Contribution

The paper provides a rare crystal structure of a halogenase with a non-native substrate, revealing the structural basis for regioselective bromination.

## Key findings

- Thal efficiently brominates tryptoline with high regioselectivity.
- Mutations G113S and G469S enhance the regioselectivity of Thal.
- The crystal structure shows how tryptoline binds in the active site of Thal.

## Abstract

Flavin‐dependent halogenases (FDHs) carry out substrate‐specific and regioselective halogenation reactions in the biosynthesis of various halogenated natural compounds. Several FDHs convert non‐native substrates in vitro. However, obtaining experimental structures of FDHs with non‐native substrates remains challenging, and docking often produces ambiguous results. Hence, there is a lack of data on how non‐native substrates bind to FDHs. Here, we show that the tryptophan 6‐halogenase Thal efficiently brominates the tricyclic indole derivative tryptoline (1,2,3,4‐tetrahydro‐β‐carboline) with high regioselectivity. The two point mutations G113S and G469S improve regioselectivity even further. A crystal structure reveals how tryptoline binds to the active site of Thal. The halogenated carbons are located close to the catalytic lysine, and the NH of tryptoline's tetrahydropyridine is positioned like the amino group of the native substrate tryptophan. The substrate binding loop of Thal is closed, again resembling the binding of tryptophan. Our work extends the range of non‐native substrates accepted by Thal, confirming the versatility of this FDH. Moreover, it is a rare example of an FDH structure in complex with a non‐native substrate.

This study shows that the flavin‐dependent tryptophan halogenase Thal also regioselectively halogenates the tricyclic non‐native substrate tryptoline, a β‐carboline with various pharmacological activities. Interestingly, Thal brominates a different position of the shared pharmacophore indole in tryptoline than it does in tryptophan. A crystal structure of Thal in complex with tryptoline explains this different regioselectivity.© 2025 WILEY‐VCH GmbH