Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

Abstract

ABSTRACTThe increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except inMycobacterium tuberculosis, where it has so far been underexploited.d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within thed-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies ofM. tuberculosis. During our studies on alternative inhibitors of thed-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinantM. tuberculosisAlr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit thed-alanine pathway in intact cells, consistent with its poorin vitroactivity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI inM. tuberculosisand thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.