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Molecules 2018, 23(2), 324; https://doi.org/10.3390/molecules23020324

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

Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China
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Received: 21 December 2017 / Revised: 23 January 2018 / Accepted: 1 February 2018 / Published: 3 February 2018
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Abstract

(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 (KmATP: 50.327 ± 4.652 μmol/L) than alanine (KmAla: 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. View Full-Text
Keywords: Mycobacterium; d-alanine-d-alanine ligase; peptidoglycan Mycobacterium; d-alanine-d-alanine ligase; peptidoglycan
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Yang, S.; Xu, Y.; Wang, Y.; Ren, F.; Li, S.; Ding, W.; Ma, Y.; Zhang, W. The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis. Molecules 2018, 23, 324.

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