Oluwole Abraham O, Hernández-Rocamora Víctor M, Cao Yihui, Li Xuechen, Vollmer Waldemar, Robinson Carol V, Bolla Jani R
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, U.K.
The Kavli Institute for Nanoscience Discovery, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
J Am Chem Soc. 2024 Mar 13;146(10):7007-7017. doi: 10.1021/jacs.4c00081. Epub 2024 Mar 1.
The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane enzymes, as well as their in situ inhibition and cofactor binding, to inform the mode of action of emerging antibiotics.
耐药病原体的迅速传播以及新抗生素发现数量的减少引发了一场全球健康危机，并激发了人们对寻找新型抗生素的浓厚兴趣。除了发现新抗生素外，阐明其作用机制需要新的方法，特别是对于那些与脂质底物和膜蛋白相互作用的抗生素。在此，我们开发了一种用于实时监测两种细菌膜磷酸酶UppP和PgpB活性的方法。然后，我们展示了如何使用杆菌肽和替考拉宁等现有及新发现的抗生素来抑制它们的活性。此外，我们发现UppP二聚体由磷脂酰乙醇胺稳定，出乎意料的是，这提高了底物加工的速度。总体而言，我们的结果证明了天然质谱在实时监测膜酶生物合成反应、原位抑制及辅因子结合方面的潜力，有助于了解新型抗生素的作用模式。
