Redirecting MurD inhibitor preference toward MurD2 through glutamate stereochemical inversion

UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) has been extensively investigated as an antibacterial target, whereas the homologous enzyme MurD2, which specifically recognizes L-glutamate, remains largely unexplored despite its restricted distribution in phytopathogenic and opportunistic bacteria. Representative MurD inhibitors retain the D-glutamate pharmacophore, which mimics the natural substrate at the glutamate-binding site, while additional substituents improve binding affinity through interactions with adjacent binding pockets. Given the opposite substrate stereoselectivities of MurD and MurD2, inversion of the glutamate stereochemistry in established MurD inhibitors represents a straightforward strategy for redirecting their inhibitory preference toward MurD2. Here, we synthesized the L-glutamate analogue of a previously reported D-glutamate-containing MurD inhibitor by replacing only the terminal glutamate stereochemistry while retaining the parent inhibitor scaffold. Biochemical analyses demonstrated that this single stereochemical inversion reversed the inhibitory preference from MurD to MurD2. Kinetic analyses indicated that both inhibitors act through substrate-mimetic inhibition by competing with UDP-N-acetylmuramoyl-L-alanine and the corresponding glutamate substrate. Structural modeling suggested that the inhibitors can occupy the substrate-binding site of XoMurD2 in a manner analogous to that observed for EcMurD, consistent with the substrate-competitive inhibition observed experimentally. These findings provide the first experimental demonstration that simple inversion of terminal glutamate stereochemistry can reverse the inhibitory preference from MurD to MurD2.