MurD is one of the ATP-driven Mur ligases that catalyze the synthesis of the UDP-MurNAcpentapeptide of the rigid cell wall for bacteria to survive, MurD is a potential target for anti-bacterial drugs. MurD catalyzes the formation of a peptide bond between the carboxyl group of the UDP-N-acetylmuramoyl- L-alanine (UMA) and the amino group of the D-glutamic acid. Though there have been reported several crystal structures of MurD in the presence and absence of its substrates, the detailed molecular mechanism is still unclear. Especially, dynamic structural transition coupled with enzymatic process needs to be unveiled. Here we report Nuclear Magnetic Resonance (NMR)-based approach to investigate the dynamic structural changes of MurD coupled with substrate binding and ATP-hydrolysis in solution, exploiting NMR titration and paramagnetic lanthanide probe method (Saio et al.). Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects in NMR spectra of the protein, such as a pseudo-contact shift (PCS). PCS is a chemical shift change induced by the paramagnetism of the lanthanide ion and provides long-range (~40 Å) distance and angular information on the observed nuclei. We introduced the lanthanide ion to MurD by the use of lanthanide chelating reagent attached to the surface of MurD via double disulfide bridges. Our NMR-based study identified a novel intermediate state of the C-terminal domain that regulates the binding of the substrates. Combined with the existing knowledge, we propose the molecular mechanism of MurD that will provide a key feature for structurebased drug design.