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.

Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host

“…The phospho-MviN-FhaA complex was interpreted to regulate late stage peptidoglycan biosynthesis, thus establishing a regulatory feedback loop that can control peptidoglycan biosynthesis (54). In addition, Mur synthetases have been shown to interact with PknA and PknB (50). Based on our findings and previous data, we think that in addition to dictating PknB localization, the PASTA domainligand interaction also regulates the activity levels of PknB (23,26).…”

An Integration of Interdisciplinary Translational Research in Anti-TB Drug Discovery: Out of the University Research Laboratories to Combat Mycobacterium tuberculosis

“…As cell division and PG biosynthesis genes share the dcw cluster, and that the PG biosynthetic protein PBP3 has been reported earlier to interact with both FtsW and FtsZ, therefore forming a trimeric complex in mycobacteria [26], it was hypothesized that an interaction between the PG synthesis and cell division proteins could exist. In addition to a confirmation to that, it was further reported that the regulation of mycobacterial Mur ligases were through protein-protein interactions with Ser/Thr Protein kinases and via phosphorylation [25,27].…”

“…Furthermore, presence of divalent cations was found to be an absolute requirement for their activities. Also higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all ATP dependent Mur ligases suggesting a stringent control of the cytoplasmic steps of peptidoglycan biosynthetic pathway in M. tuberculosis [23][24][25].…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…Dr Bhakta (Birkbeck, University of London, UK) has pioneered research in the metabolism of peptidoglycan (PG) in M. tuberculosis for over a decade. 123 The ATP dependent Mur ligase genes are involved in the synthesis of PG precursors. These genes are restricted to bacteria and have been found to be essential in mycobacteria by transposon site hybridization (TraSH).…”