Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
Org Biomol Chem. 2019 May 15;17(19):4720-4724. doi: 10.1039/c8ob03229g.
Metabolic engineering of fatty acids and polyketides remains challenging due to unresolved protein-protein interactions that are essential to synthase activity. While several chemical probes have been developed to capture and visualize protein interfaces in these systems, acyl carrier protein (ACP) transacylase (AT) domains remain elusive. Herein, we combine a mutational strategy with fluorescent probe design to expedite the study of AT domains from fatty acid and polyketide synthases. We describe the design and evaluation of inhibitor-inspired and substrate-mimetic reporters containing sulfonyl fluoride and β-lactone warheads. Moreover, specific active-site labeling occurs by optimizing pH, time, and probe concentration, and selective labeling is achieved in the presence of inhibitors of competing domains. These findings provide a panel of AT-targeting probes and set the stage for future combinatorial biosynthetic and drug discovery initiatives.
由于尚未解决的蛋白质-蛋白质相互作用对于合成酶活性至关重要，脂肪酸和聚酮的代谢工程仍然具有挑战性。虽然已经开发了几种化学探针来捕获和可视化这些系统中的蛋白质界面，但酰基辅酶 A（ACP）转酰基酶（AT）结构域仍然难以捉摸。在这里，我们将突变策略与荧光探针设计相结合，以加速脂肪酸和聚酮合酶的 AT 结构域的研究。我们描述了含有磺酰氟和β-内酰胺弹头的抑制剂启发和底物模拟报告子的设计和评估。此外，通过优化 pH 值、时间和探针浓度来实现特异性活性位点标记，并且在存在竞争结构域抑制剂的情况下可以实现选择性标记。这些发现提供了一组针对 AT 的靶向探针，并为未来的组合生物合成和药物发现计划奠定了基础。
