Chemical proteomics enhances the understanding of 2AA stress in Salmonella enterica

ABSTRACT Pyridoxal 5′-phosphate (PLP) is an essential cofactor for enzymes that catalyze diverse reactions in central metabolism. 2-Aminoacrylate (2AA) is a reactive enamine and an obligate catalytic intermediate in some PLP-mediated reactions. In the absence of the enamine/imine deaminase RidA, Salmonella enterica accumulates 2AA, which causes cellular stress. 2AA can attack PLP in the active site of some enzymes and covalently inactivate them by forming a 2AA-PLP adduct, which has already been characterized for some target enzymes in vivo and in vitro. The mechanism of 2AA attack suggests that a majority of cellular PLP-DEs would be targets of 2AA damage. Herein, a chemical proteomics workflow that uses PL (pyridoxal) probes to enrich PLP-DEs with a click chemistry-based protocol was implemented to investigate the global scale of 2AA damage in S. enterica. The results showed that PLP-DEs could be enriched in S. enterica with two different PL probes. When cells were labeled by providing a PL probe as the sole source of vitamin B6, several proteins were found to be more enriched when grown in conditions of high 2AA versus low 2AA stress. These data identified proteins that were previously shown to be attacked by 2AA as well as new candidate targets, demonstrating the usefulness of this approach to define the 2AA stress response with a global perspective. Growth analyses indicated that 2AA stress impacts the salvage of PL probes, suggesting that these and other PL probes will be valuable in future physiological studies to understand PLP salvage, a critical pathway in all organisms. In total, this study expands our understanding of 2AA metabolism and takes an initial step toward characterizing the global impact of 2AA stress in S. enterica.IMPORTANCELoss of RidA homologs results in 2-aminoacrylate stress in Salmonella and other bacteria. The stress is derived from the reaction of 2AA with a pyridoxal phosphate cofactor in metabolic enzymes, which inactivates the respective enzymes. This study uses a chemical proteomic method and, with an initial test case, explores the damage that is generated by 2AA on a global proteomic scale. This work provides a basis for probing the extent of 2AA stress in different organisms and for identifying the enzymes targeted by 2AA.