1. Inhibition of bacterial growth by beta-chloro-D-alanine
J M Manning, N E Merrifield, W M Jones, E C Gotschlich Proc Natl Acad Sci U S A. 1974 Feb;71(2):417-21. doi: 10.1073/pnas.71.2.417.
The D- and L-isomers of beta-chloroalanine inhibit the growth of Diplococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, and Escherichia coli. With pneumococcus the inhibition by beta-chloro-D-alanine is completely prevented by either D-alanine or D-alanyl-D-alanine, while L-alanine is not effective in preventing the inhibition. The inhibition of growth by beta-chloro-L-alanine is not affected by D-alanine and is only partially prevented by high concentrations of L-alanine. The intracellular free alanine in untreated E. coli and B. subtilis is about 95% in the D-configuration while the free intracellular alanine in both organisms after treatment with beta-chloro-D-alanine is predominantly the L-isomer. These results suggested that the beta-chloroamino acid inactivates alanine racemase (EC 5.1.1.1). Indeed, when extracts of E. coli or B. subtilis were treated with beta-chloro-D-alanine, the activities of alanine racemase and of D-glutamate-D-alanine transaminase were found to be 90-95% inhibited. Studies with mice have shown that beta-chloro-D-alanine is an effective antibacterial agent in vivo againt D. pneumoniae, S. pyogenes, and E. coli.
2. Coupling of alanine racemase and D-alanine dehydrogenase to active transport of amino acids in Escherichia coli B membrane vesicles
G Kaczorowski, L Shaw, M F-entes, C Walsh J Biol Chem. 1975 Apr 25;250(8):2855-65.
Isolated membrane vesicles from Escherichia coli B grown on DL-alanine-glycerol carry out amino acid active transport coupled to D-alanine oxidation by a membrane-bound dehydrogenase. Several other D-amino acids are substrates for this D-alanine dehydrogenase and also drive concentrative uptake of solutes. Additionally, L-alanine and L-serine can energize solute transport by virtue of conversion to oxidizable D isomers by a membrane-bound alanine racemase. No other physiological L-amino acids were effective. Both membrane enzymes and consequent solute transport are markedly reduced in vesicles from glucose-grown cells. Respiratory chain uncouplers abolish the racemase-dehydrogenase-supported transport activity. When amino-oxyacetate at 10-4 M is added to the vesicles, the racemase activity and transport driven by L-alanine and L-serine is specifically and reversibly inhibited. D-Alanine-driven transport is unaffected. Similarly beta-chloro-L-alanine is an irreversible inactivator of the bound racemase but not the D-alanine dehydrogenase. Both the D and L isomers of beta-chloroalanine support oxygen uptake by the vesicles and initially stimulate L-(14C)proline active transport. However, oxidation of the beta-chloro-D-alanine rapidly uncouples active transport from substrate oxidation. This transport inactivation can be protected partially by dithiothreitol, putatively scavenging a reactive product of chloroalanine oxidation. Authentic beta-chloropyruvate produces the same transport uncoupling. When beta-chloro-L-alanine is employed as a substrate, no such transport inactivation is observed. This difference may stem from the possibility that the alanine racemase eliminates HCl from beta-chloro-L-alanine producing pyruvate, not the beta-chloropyruvate that would arise from racemization and then dehydrogenation. We have shown that exogenous pyruvate is oxidized by the vesicles and will also stimulate active transport of amino acids.
3. Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis
Gareth A Prosser, Anne Rodenburg, Hania Khoury, Cesira de Chiara, Steve Howell, Ambrosius P Snijders, Luiz Pedro S de Carvalho Antimicrob Agents Chemother. 2016 Sep 23;60(10):6091-9. doi: 10.1128/AAC.01249-16. Print 2016 Oct.
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.
