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.
Highlights
Tuberculosis (TB) is a one of the leading causes of human mortality from infectious diseases with an estimated 1.4 million deaths globally in 2011 [1]
Cloning of M. tuberculosis genes The murC (Rv2152c) and murF (Rv2157c) genes were amplified from M. tuberculosis H37Rv genomic DNA using Phusion hot start DNA polymerase and primers listed in table S1, and cloned into pET28(b)+ vector at NdeI/BamHI sites to obtain pSBC2 and pSBC4 respectively. pVLT31, derived from pMMB207, does not encode for a fusion-tag [22]; pSBC1 [15], pSBC2 and pSBC4 were digested with XbaI/HindIII to give,2.0 kb fragments containing the ribosome binding site (RBS), His-tag, a thrombin cleavage site and the genes of interest, which were sub-cloned into pVLT31 at the same sites to obtain p31E, p31C and p31F respectively
The purity obtained for each protein using P. putida as a host for over-expression was considerably higher (,95%) in comparison to that achieved with E. coli (,85%)
Summary
Tuberculosis (TB) is a one of the leading causes of human mortality from infectious diseases with an estimated 1.4 million deaths globally in 2011 [1]. Control of TB has become much harder with the recent emergence of extensively-drug resistant TB (XDR-TB) strains, as there is virtually no effective drug available for their treatment [2]. New drugs with novel mechanisms of action are urgently required to tackle the spread of drug-resistant TB strains. Mycobacterium tuberculosis, the causative pathogen of TB, is extremely tolerant to chemical agents and this feature is attributed to its remarkably impermeable cell wall, which consists of a covalently linked mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. To date not a single clinically available drug has been reported to target ATP-dependent Mur ligases (Mur synthetases), which are key enzymes of the PG biosynthetic pathway. Our findings on the inhibition of MurE synthetase in M. tuberculosis have highlighted this group of enzymes as potential antimycobacterial targets [5,6,7]
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