d-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition

Abstract

Summary 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 analogue 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.

Metabolomics reveals that the cAMP receptor protein regulates nitrogen and peptidoglycan synthesis in Mycobacterium tuberculosis

“…33 and 34 ) or the cAMP analogue, dibutyryl-cAMP, analogue which is less hydrophobic than cAMP and is therefore able to pass through cell membranes, where it accumulates intracellularly as mono-butyryl-cAMP. 35 – 37 Further, as one of the pathway is the synthesis of the precursor of peptidoglycan, one could investigate the antimicrobial susceptibility of M. tuberculosis Δc rp to antimicrobials such as d -cycloserine which targets alanine racemase and d -alanine ligase 38 and vancomycin which binds the d -Ala- d -Ala terminal of the growing peptide chain. 39 Based on the data presented here, we can anticipate an increase in susceptibility of M. tuberculosis Δc rp compared to the parental strain.…”

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition

“…231,232 An important inhibitor of alanine racemase is D-cycloserine, a natural product used in the treatment of drug resistant tuberculosis. 233,234 It is notable that the biosynthetic pathway for D-cycloserine contains a reaction catalysed by a racemase, O-ureidoserine racemase 46 (vide supra, Scheme 10A). D-Cycloserine 93 is a relatively non-specific antibiotic and targets M. tuberculosis alanine racemase and D-Ala-D-Ala ligase.…”

Cell Surface Biosynthesis and Remodeling Pathways in Mycobacteria Reveal New Drug Targets

“…As there are significant similarities in the structures of MurC and MurD/ MurE and MurF, it should be possible to design inhibitors that can inhibit the Mur enzymes simultaneously (Kouidmi et al, 2014). Still within the cytoplasm, alanine racemase (Alr) catalyzes the conversion of L-alanine to D-alanine which is required to synthesize the peptide component of Lipid II (De Chiara et al, 2020). There are currently several commercial antibiotics targeting Alr (see Table 1), including D-cycloserine (Kim et al, 2003).…”

“…D-Cycloserine has been used to treat pulmonary and extra-pulmonary TB, including MDR-TB, but this is hampered by severe toxic side-effects (Azam et al, 2016). Recently, it was shown that Alr activity remains detectable in Mtb exposed to clinically relevant D-cycloserine concentrations (Prosser and De Carvalho, 2013;De Chiara et al, 2020). This is due to reversible binding via D-cycloserine-adduct hydrolysis thus enabling dissociation and structural rearrangement within the active enzyme to regain activity.…”

“…This is due to reversible binding via D-cycloserine-adduct hydrolysis thus enabling dissociation and structural rearrangement within the active enzyme to regain activity. This mechanistic insight now provides a route for discovery of improved Alr inhibitors (De Chiara et al, 2020). D-cycloserine also inhibits D-Ala:D-Ala ligase (DdlA), another essential enzyme in PG biosynthesis (Prosser and De Carvalho, 2013) and improvements on the activity of this compound may yield increased activity to DdlA also.…”