Enzymes are powerful catalysts for site-selective C–H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence–function information on flavin-dependent halogenases (FDHs). This broad survey provided a number of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-molecule substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds were characterized and used for preparative-scale late-stage C–H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution.

中文翻译：

---

通过家庭范围的活性谱鉴定，使用黄素依赖性卤化酶进行站点选择性CH卤化

酶是位点选择性C–H键功能化的强大催化剂。然而，由于从序列信息预测催化活性的基本困难，鉴定用于该任务和用于生物催化的合适的酶通常仍然具有挑战性。在这项研究中，整个家庭的活动图谱用于获得黄素依赖性卤化酶（FDHs）的序列功能信息。这项广泛的调查提供了许多有关FDH活性的见解，包括卤化物的特异性和底物的偏爱性，这些见解从迄今报道的更加集中的研究中还不明显。还确定了FDH序列空间中最可能包含适合于卤化小分子底物的酶的区域。具有新型底物范围和大区域互补区域选择性的FDH 对三维复杂化合物进行了表征，并用于制备规模的后期C–H功能化。在许多情况下，这些酶提供的活性需要在之前的工作中完成几轮定向进化才能完成，从而突出表明该方法可以节省大量时间来鉴定生物催化剂，并为进一步进化提供了先进的起点。