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ATP-dependent MurE ligase in Mycobacterium tuberculosis: Biochemical and structural characterisation

DOI: 10.1016/j.tube.2009.10.007 DOI Help
PMID: 19945347 PMID Help

Authors: Chandrakala Basavannacharya (Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London) , Giles Robertson (Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London) , Tulika Munshi (Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London) , Sanjib Bhakta (Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London) , Nicholas Keep (Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Tuberculosis , VOL 90 (1) , PAGES 16 - 24

State: Published (Approved)
Published: January 2010

Abstract: New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0 Å resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.

Journal Keywords: Bacterial; Cell; Cloning; Molecular; Crystallography; X-Ray; Humans; Mycobacterium; Peptide; Peptidoglycan; Tuberculosis; Up-Regulation

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Added On: 02/02/2010 16:32

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