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

被引：44

作者：

Basavannacharya, Chandrakala ^{[1
]}

Robertson, Giles ^{[1
]}

Munshi, Tulika ^{[1
]}

Keep, Nicholas H. ^{[1
]}

Bhakta, Sanjib ^{[1
]}

机构：

[1] Univ London, Inst Struct & Mol Biol, Dept Biol Sci, London WC1E 7HX, England

来源：

TUBERCULOSIS | 2010年 / 90卷 / 01期

基金：

英国生物技术与生命科学研究理事会;

关键词：

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;

D O I：

10.1016/j.tube.2009.10.007

中图分类号：

R392 [医学免疫学]; Q939.91 [免疫学];

学科分类号：

100102 ;

摘要：

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.

引用

页码：16 / 24

页数：9

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