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Instruments / Technical Area	Publication Details	Published	Actions
I04-Macromolecular Crystallography	Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target Sudagar S. Gurcha , Veeraraghavan Usha , Jonathan A. G. Cox , Klaus Futterer , K Abrahams , Apoorva Bhatt , Luke Alderwick , Robert C. Reynolds , Nicholas J. Loman , Vijayashankar Nataraj , Carlos Alemparte , David Barros , Adrian J. Lloyd , Lluis Ballell , Judith V. Hobrath , Gurdyal S. Besra Plos One 9 (11) DOI: 10.1371/journal.pone.0113568 PMID: 25409504 Diamond Proposal Number(s): [10369] Open Access Show Abstract ▼ Abstract: The 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 10× 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 Å.	Nov 2014

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I04-Macromolecular Crystallography

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target