ATP-dependent MurE ligase in Mycobacterium tuberculosis: Biochemical and structural characterisation.
16 - 24.
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.
|Title:||ATP-dependent MurE ligase in Mycobacterium tuberculosis: Biochemical and structural characterisation|
|Keywords:||Cell wall peptidoglycan biosynthesis, ATP-dependent ligase, Tuberculosis, Enzyme kinetics, Protein structure, ALANYL-D-GLUTAMATE, BACTERIAL URIDINE NUCLEOTIDES, ACID-ADDING ENZYME, COMPLETE GENOME SEQUENCE, CELL WALL BIOSYNTHESIS, L-LYSINE LIGASE, ESCHERICHIA-COLI, PEPTIDOGLYCAN BIOSYNTHESIS, PHOSPHINATE INHIBITORS, STAPHYLOCOCCUS-AUREUS|
|UCL classification:||UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of)
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology
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