ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation.
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 3.0A 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.
Basavannacharya
Chandrakala
C
Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK.
Robertson
Giles
G
Munshi
Tulika
T
Keep
Nicholas H
NH
Bhakta
Sanjib
S
D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis / Q000235:genetics*; D014376:Tuberculosis / Q000201:enzymology / Q000378:metabolism*; D015854:Up-Regulation
D001426:Bacterial Proteins; D010457:Peptidoglycan; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C554750:MurE protein, Mycobacterium tuberculosis