Shepherd Sarah A, Menon Binuraj R K, Fisk Heidi, Struck Anna-Winona, Levy Colin, Leys David, Micklefield Jason
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
Chembiochem. 2016 May 3;17(9):821-4. doi: 10.1002/cbic.201600051. Epub 2016 Mar 30.
Flavin-dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X-ray crystal structure of a tryptophan 6-halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure-guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3-indolepropionate 75 % 5-chlorination was observed with the mutant in comparison to 90 % 6-chlorination for the wild-type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non-indolic substrates.
黄素依赖性卤化酶是用于芳香族化合物区域选择性卤化的潜在有用生物催化剂。卤代芳香族化合物可用于药物和其他有价值产品的合成与生物合成。在此，我们报道了色氨酸6 - 卤化酶（SttH）的首个X射线晶体结构，这使得能够鉴定出色氨酸卤化酶中对区域选择性有贡献的关键残基。基于结构的诱变产生了一个三重突变体（L460F/P461E/P462T），其区域选择性发生了完全转变；对于底物3 - 吲哚丙酸，与野生型SttH的90% 6 - 氯化相比，该突变体观察到75%的5 - 氯化。这是如何从单一亲本酶创建区域互补卤化酶的首个明确示例。SttH的生物催化范围也得以扩展，包括一系列吲哚类和非吲哚类底物。
