title: Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis.
abstract: 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.
authors.affiliation: Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London, United Kingdom Luiz.Carvalho@crick.ac.uk gareth.prosser@nih.gov.
authors.forename: Gareth A
authors.initials: GA
authors.lastname: Prosser
authors.affiliation: Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Rodenburg
authors.affiliation: Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London, United Kingdom.
authors.forename: Hania
authors.initials: H
authors.lastname: Khoury
authors.affiliation: Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London, United Kingdom.
authors.forename: Cesira
authors.initials: C
authors.lastname: de Chiara
authors.affiliation: Proteomics Scientific Technology Platform, Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom.
authors.forename: Steve
authors.initials: S
authors.lastname: Howell
authors.affiliation: Proteomics Scientific Technology Platform, Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom.
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.affiliation: Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London, United Kingdom Luiz.Carvalho@crick.ac.uk gareth.prosser@nih.gov.
authors.forename: Luiz Pedro S
authors.identifier: 0000-0003-2875-4552
authors.initials: LP
authors.lastname: de Carvalho
mesh_terms: D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010457:Peptidoglycan / Q000096:biosynthesis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010457:Peptidoglycan; D011994:Recombinant Proteins; D015091:beta-Alanine; C006977:3-chloroalanine; D000591:Amino Acid Isomerases; C062292:glutamate racemase