Bork Simon, Besse Caroline, Sewald Norbert, Niemann Hartmut H
Department of Chemistry, Structural Biochemistry, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Chembiochem. 2025 Jul 11;26(13):e202500246. doi: 10.1002/cbic.202500246. Epub 2025 Jun 17.
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.
黄素依赖性卤化酶（FDHs）在各种卤代天然化合物的生物合成中进行底物特异性和区域选择性卤化反应。几种FDHs在体外可转化非天然底物。然而，获得与非天然底物结合的FDHs的实验结构仍然具有挑战性，并且对接往往会产生不明确的结果。因此，缺乏关于非天然底物如何与FDHs结合的数据。在此，我们表明色氨酸6-卤化酶Thal能以高区域选择性高效地将三环吲哚衍生物色醇（1,2,3,4-四氢-β-咔啉）溴化。两个点突变G113S和G469S进一步提高了区域选择性。晶体结构揭示了色醇如何与Thal的活性位点结合。卤化碳靠近催化赖氨酸，色醇四氢吡啶的NH的位置类似于天然底物色氨酸的氨基。Thal的底物结合环是闭合的，这再次类似于色氨酸的结合。我们的工作扩展了Thal所接受的非天然底物的范围，证实了这种FDH的多功能性。此外，这是FDH与非天然底物形成复合物的结构的罕见实例。
