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 Mycobacterium tuberculosis (Mtb), 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 Tb-DdlA. (2) Methods and Results: Rv2981c (ddlA) was expressed in Escherichia coli, and the purified Tb-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 Tb-DdlA indicated that Tb-DdlA exhibited a higher affinity to ATP (K-mATP: 50.327 +/- 4.652 +/- mol/L) than alanine (K-mAla: 1.011 +/- 0.094 mmol/L). A colorimetric assay for Tb-DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on Tb-DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of Tb-DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring Tb-DdlA biology, and developed an easy colorimetric assay for screening of Tb-DdlA inhibitors.