The 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 in Mycobacterium tuberculosis, where it has so far been underexploited. d-
Cycloserine, a clinically approved antituberculosis therapeutic, inhibits
enzymes within the d-
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 of M.
tuberculosis During our studies on alternative inhibitors of the d-
alanine pathway, we discovered that the canonical
alanine racemase (Alr) inhibitor β-chloro-d-
alanine (
BCDA) is a very poor inhibitor of recombinant M.
tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that
BCDA does not inhibit the d-
alanine pathway in intact cells, consistent with its poor in vitro activity, 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 in M.
tuberculosis and thus provides a valuable tool for studying this essential and enigmatic
enzyme and a starting point for future MurI-targeted antibacterial development.