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Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target
Structural highlights
FunctionSYDND_MYCS2 Aspartyl-tRNA synthetase with relaxed tRNA specificity since it is able to aspartylate not only its cognate tRNA(Asp) but also tRNA(Asn). Reaction proceeds in two steps: aspartate is first activated by ATP to form Asp-AMP and then transferred to the acceptor end of tRNA(Asp/Asn).[HAMAP-Rule:MF_00044] Publication Abstract from PubMedThe human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10x the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 A. Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target.,Gurcha SS, Usha V, Cox JA, Futterer K, Abrahams KA, Bhatt A, Alderwick LJ, Reynolds RC, Loman NJ, Nataraj V, Alemparte C, Barros D, Lloyd AJ, Ballell L, Hobrath JV, Besra GS PLoS One. 2014 Nov 19;9(11):e113568. doi: 10.1371/journal.pone.0113568., eCollection 2014. PMID:25409504[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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