Real-Time Biosynthetic Reaction Monitoring Informs the Mechanism of Action of Antibiotics

\ua9 2024 The Authors. Published by American Chemical SocietyThe 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

Structural basis for the hydrolytic activity of the transpeptidase-like protein DpaA to detach Braun’s lipoprotein from peptidoglycan

\ua9 2023 Wang et al.The peptidoglycan layer is a defining characteristic of bacterial cells, providing them with structural support and osmotic protection. In Escherichia coli, this layer is linked to the outer membrane via the abundant membrane-anchored protein Lpp, known as Braun’s lipoprotein, with LD-transpeptidases LdtA, LdtB, and LdtC catalyzing the attachment. However, one distinctive member of the YkuD-type transpeptidase family, LdtF (recently renamed DpaA), carries out the opposite reaction of detaching Lpp from the peptidoglycan layer. In this study, we report the crystal structure of DpaA, which reveals the enzyme’s ability to cleave, rather than form, the Lpp-peptidoglycan linkage. Assays with purified peptidoglycan-Lpp as the substrate and chemically synthesized compounds suggest that DpaA’s shallow L-shaped active site can only accommodate and cleave the peptidoglycan-Lpp cross-link with a constrained conformation. This study provides insights into how homologous Ldt enzymes can perform opposing chemical reactions