From the Structural Biochemistry and.
Organic and Bioorganic Chemistry Research Groups, Department of Chemistry, Bielefeld University, Universitätsstrasse 25, D-33615 Bielefeld, Germany.
J Biol Chem. 2019 Feb 15;294(7):2529-2542. doi: 10.1074/jbc.RA118.005393. Epub 2018 Dec 17.
Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH, oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate trypto-phan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ϵ-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of l-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.
黄素依赖的卤化酶作为有机合成中的生物催化剂越来越受到关注，它们促进了只需要 FADH、氧气和卤盐的环保卤化策略。不同的黄素依赖色氨酸卤化酶选择性地在 C5、C6 或 C7 位氯化或溴化色氨酸的吲哚部分。在这里，我们以 2.55Å 的分辨率呈现了第一个色氨酸 6-卤化酶，即来自细菌的 Thal（也称为 ThdH）的底物结合结构。该结构表明，色氨酸的 C6 位于保守赖氨酸的 ε-氨基旁边，证实了这样的假设，即与催化残基的接近程度决定了亲电芳香取代的位置。尽管 Thal 在序列和结构上与色氨酸 7-卤化酶 RebH 更相似，而不是与色氨酸 5-卤化酶 PyrH，但 Thal 活性位点中的吲哚结合构象更类似于 PyrH，而不是 RebH。Thal 和 RebH 之间吲哚取向的差异似乎主要由定位色氨酸骨架原子的残基决定。Thal 和 RebH 排列在底物结合位点的序列仅在少数残基上有所不同。因此，我们用 RebH 中的对应残基替换了 Thal 活性位点中的五个氨基酸，生成了五重变体 Thal-RebH5。总体上，Thal-RebH5 变体对 l-Trp 的转化类似于 WT Thal，但它的氯化和溴化的区域选择性几乎完全从 C6 切换到 RebH 中的 C7。我们得出结论，基于结构的蛋白质工程通过有针对性的取代少数残基是一种有效的方法，可以定制黄素依赖的卤化酶。
