The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis.

(1) Background: d-alanine-d-alanine ligase (DdlA), an effective target for drug development to combat against (), which threatens human health globally, supplies a substrate of d-alanyl-d-alanine for peptidoglycan crosslinking by catalyzing the dimerization of two d-alanines. To obtain a better understanding of DdlA profiles and develop a colorimetric assay for high-throughput inhibitor screening, we focused on explicating and characterizing -DdlA. (2) Methods and Results: () was expressed in , and the purified -DdlA was identified using (anti)-polyhistidine antibody followed by DdlA activity confirmation by measuring the released orthophosphate via colorimetric assay and the yielded d-alanyl-d-alanine through high performance thin layer chromatography (HP-TLC). The kinetic assays on -DdlA indicated that -DdlA exhibited a higher affinity to ATP (K: 50.327 ± 4.652 μmol/L) than alanine (K: 1.011 ± 0.094 mmol/L). A colorimetric assay for -DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on -DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of -DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring -DdlA biology, and developed an easy colorimetric assay for screening of -DdlA inhibitors.