Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis

Abstract

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Figure 1. Analysis of purified recombinant M. tuberculosis Mur synthetases.

(A) SDS-PAGE analysis of MurC (lane 1), MurD (lane 2), MurE (lane 3) and MurF (lane 4) with protein molecular weight markers (lane 5) and (B) specific activity of each protein. Error bar at 1 SD based on assays conducted in triplicate for each protein.

Figure 2. Determination of substrate specificities of Mur synthetases.

Different (A) Nucleotides (B) Amino acids (C) Uridine sugars and (D) divalent and monovalent cations (at 5 mM concentration) were tested to analyze their specificities for MurC, MurD and MurF synthetases. X-axis represents different substrates used. Y-axis, in all the cases, represents the amount of P_i released in pmol/min.

Figure 3. Estimation of optimal substrate concentration for Mur synthetases.

Inhibition curves obtained for MurC, MurD and MurF synthetases with (A) ATP and (B) their respective uridine sugars. X-axis represents substrate concentration used and Y-axis is the percent inhibition calculated for each concentration.

Figure 4. dcw operon analysis.

(A) Representation of the M. tuberculosis genomic region (2408385–2424838), showing ORFs and gaps, and highlighting regions upstream of the dcw operon screened for the presence of a promoter driving the operon. (B) cDNA analysis for identifying boundaries of the dcw operon. (C) Promoter analysis by cloning into the promoter-less vector pYUB76 and β-galactosidase assay for confirmation of promoter activity.

Figure 5. Protein-protein interaction studies of M. tuberculosis Mur synthetases.

(A) Interaction using an M-PFC where growth on TMP plates at 12.5 µg/mL concentration indicated a positive protein-protein interaction, (B) Quantitation of M-PFC interactions by the resazurin assay and (C) representation of final interaction results. Each interaction, by both methods, was assayed in triplicate.