The broad-spectrum antibiotic d-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of d-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and d-Ala:d-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.
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Crystallographic atomic coordinates and structure factors have been deposited in the PDB under accession code 6SCZ. The data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank V.L. Schramm, R. Guimarães da Silva, C. Melo Czekster and S.R. Lovell-Read for critical reading of the manuscript and P. Walker for help with crystallographic data acquisition at an early stage of the project. Work in the Mycobacterial Metabolism and Antibiotic Research Laboratory was chiefly supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (grant no. FC001060), the UK Medical Research Council (grant no. FC001060), the Wellcome Trust (grant no. FC001060) and also by a Wellcome Trust New Investigator Award 104785/B/14/Z (to L.P.S.C). NMR data were recorded at the MRC Biomedical NMR Centre at the Francis Crick Institute, which receives core funding from Cancer Research UK (grant no. FC001029), the Medical Research Council (grant no. FC001029) and the Wellcome Trust (grant no. FC001029). We acknowledge I04–1 beamline of the Diamond Light Source Synchrotron (Oxford, UK, mx13775-39).
The authors declare no competing interests.
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de Chiara, C., Homšak, M., Prosser, G.A. et al. d-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0498-9