Active Site-Directed Probes for targeting Bacterial Phosphoarginine Phosphatases

Canonical protein phosphorylation patterns are a thoroughly studied post-translational modification (PTM) driving distinct regulatory mechanisms in both prokaryotes, and eukaryotes. In contrast, the identification and investigation of essential components that regulate non-canonical phosphorylation has received considerably less attention, although these PTMs are associated with important functions. One notable example is arginine phosphorylation which modulates processes such as protein degradation, transcriptional regulation and spore germination in bacteria. Herein we introduce the first in class covalent activity-based probes to study phosphoarginine-phosphatases. We identify unsaturated phosphonamidic acids as bespoke electrophilic phosphoarginine (pArg) mimics, which allowed to uncover a series of unprecedented pArg-phosphatases, which in part had been previously annotated as low molecular weight tyrosine-phosphatases across phylogenetically distinct microbial species. This work, which serves as the first example of proteome-wide activity-based profiling of pArg phosphatases will help inform the development of new therapeutic modalities and expand our understanding of bacterial signal transduction.