Reinterpreting the Mechanism of Inhibition of Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase by D-Cycloserine

D-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of D-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show. that D-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates D-cycloserine binds exclusively to the C-terminal D-alanine binding site, even in the absence of bound D-alanine at the N-terminal binding site. Together, these results led us to propose a new model of D-alanine:D-alanine ligase inhibition by D-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.