Insights into the Inhibition of d-Alanine-d-Alanine Ligase by Integration of Kinetics and Structural Analysis.

, a pathogenic bacterium, is harmful to humans, domestic animals, and fishes and, moreover, of public health concern due to the emergence of multiple drug-resistant strains. The cell wall has been discovered as a novel and efficient drug target against bacteria, and d-alanine-d-alanine ligase (Ddl) is considered as an essential enzyme in bacterial cell wall biosynthesis. Herein, we studied the HBNUAh01 Ddl (Ddl) enzyme activity and kinetics and determined the crystal structure of Ddl/d-Ala complex at 2.7 Å resolution. An enzymatic assay showed that Ddl exhibited higher affinity to ATP (: 54.1 ± 9.1 μM) compared to d-alanine (: 1.01 ± 0.19 mM). The kinetic studies indicated a competitive inhibition of Ddl by d-cycloserine (DCS), with an inhibition constant () of 120 μM and the 50% inhibitory concentrations (IC) value of 0.5 mM. Meanwhile, structural analysis indicated that the Ddl/d-Ala complex structure adopted a semi-closed conformation form, and the active site was extremely conserved. Noteworthy is that the substrate d-Ala occupied the second d-Ala position, not the first d-Ala position. These results provided more insights for understanding the details of the catalytic mechanism and resources for the development of novel drugs against the diseases caused by .