Tailored Pyridoxal Probes Unravel Novel Cofactor‐Dependent Targets and Antibiotic Hits in Critical Bacterial Pathogens

Unprecedented bacterial targets are urgently needed to overcome the resistance crisis. Herein we systematically mine pyridoxal phosphate‐dependent enzymes (PLP‐DEs) in bacteria to focus on a target class which is involved in crucial metabolic processes. For this, we tailored eight pyridoxal (PL) probes bearing modifications at various positions. Overall, the probes exceeded the performance of a previous generation and provided a detailed map of PLP‐DEs in clinically relevant pathogens including challenging Gram‐negative strains. Putative PLP‐DEs with unknown function were exemplarily characterized via in‐depth enzymatic assays. Finally, we screened a panel of PLP binders for antibiotic activity and unravelled the targets of hit molecules. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP‐dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP‐DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.