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

Basavannacharya, Chandrakala and Robertson, Giles and Munshi, T. and Keep, Nicholas H. and Bhakta, Sanjib (2010) ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation. Tuberculosis 90 (1), pp. 16-24. ISSN 1472-9792.

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Official URL: http://dx.doi.org/10.1016/j.tube.2009.10.007

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.

Item Type: Article
Additional Information: *Author Robertson left BBK 31.12.10, no staff ID (PR 24.2.11)
School or Research Centre: Birkbeck Schools and Research Centres > School of Science > Biological Sciences
Depositing User: Administrator
Date Deposited: 04 Aug 2010 14:09
Last Modified: 17 Apr 2013 12:17
URI: http://eprints.bbk.ac.uk/id/eprint/1026

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