Metabolomics analysis identifies d-Alanine-d-Alanine ligase as the primary lethal target of d-Cycloserine in mycobacteria.

d-Cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR) drug resistant strains of Mycobacterium tuberculosis . d-Cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of alanine racemase (Alr) and d-alanine-d-alanine ligase (Ddl). Although the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of d-alanyl-d-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of d-alanine, thus allowing DCS to outcompete d-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce d-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.