期刊
TUBERCULOSIS
卷 90, 期 1, 页码 16-24出版社
CHURCHILL LIVINGSTONE
DOI: 10.1016/j.tube.2009.10.007
关键词
Cell wall peptidoglycan biosynthesis; ATP-dependent ligase; Tuberculosis; Enzyme kinetics; Protein structure
资金
- Birkbeck Faculty
- University of London
- MRC [G0801956]
- ORS/BORS
- Birkbeck
- Biotechnology and Biological Sciences Research Council [BB/F011148/1] Funding Source: researchfish
- Medical Research Council [G0801956, G0802079] Funding Source: researchfish
- BBSRC [BB/F011148/1] Funding Source: UKRI
- MRC [G0802079] Funding Source: UKRI
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 angstrom 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. (C) 2009 Elsevier Ltd. All rights reserved.
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