Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

Abstract

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K(m) values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.

Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

“…Mahapatra et al (2000) reported that MurC was able to incorporate glycine (Gly) and L-Ala to UDP-MurNAc in both M. tuberculosis and M. leprae [13]. Our investigation of the structural and functional characterization of MurE from M. tuberculosis [14], [15] revealed that MurE was only active in the presence of its specific natural substrates: uridine-diphospho- N -acetylmuramoyl-L-alanine-D-glutamate (UDP-MurNAc-L-Ala-D-Glu), m -DAP and ATP. In this study, we comprehensively examine and compare the activity of all Mur synthetases in M. tuberculosis with respect to their natural substrates.…”

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

“…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].…”

“…Two crystal structures of Mtb-MurE have been solved with different substrates bound to it, and essential residues have been determined for this enzyme by mutation studies [23,24]. A number of initial hits against the Mtb-MurE enzyme have been reported by us recently [28][29][30] and some of these agents are also antibacterial against whole-cell M. tuberculosis, are now part of medicinal chemistry projects to optimize these hits.…”

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

“…The structure of murE has been resolved to 3A and shows an accessible active pocket site that can be exploited for drug design. 124,125 Enzymatic inhibition of MurE from M. tuberculosis was observed with the extracts from Columbian plants and chemically synthesized quinolone compounds thereby demonstrating that the ligases can serve as drug targets. [126][127][128][129] Additionally, as cell division and cell wall biogenesis are stringently controlled, co-operative processes, it is no surprise to find the mur ligase genes clustered in the division cell-wall (dcw) operon, further indicating that targeting this pathway will have knock-on effects on co-regulated pathways.…”