Functional characterization by genetic complementation of aroB-encoded dehydroquinate synthase from Mycobacterium tuberculosis H37Rv and its heterologous expression and purification

de Mendonqa, Jordana Dutra, Ely, Fernanda, Palma, Mario Sergio, Frazzon, Jeverson, Basso, Luiz Augusto and Santos, Digenes Santiago (2007) Functional characterization by genetic complementation of aroB-encoded dehydroquinate synthase from Mycobacterium tuberculosis H37Rv and its heterologous expression and purification. Journal of Bacteriology, 189 17: 6246-6252. doi:10.1128/JB.00425-07

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Author de Mendonqa, Jordana Dutra
Ely, Fernanda
Palma, Mario Sergio
Frazzon, Jeverson
Basso, Luiz Augusto
Santos, Digenes Santiago
Title Functional characterization by genetic complementation of aroB-encoded dehydroquinate synthase from Mycobacterium tuberculosis H37Rv and its heterologous expression and purification
Formatted title
Functional characterization by genetic complementation of aroB-encoded dehydroquinate synthase from Mycobacterium tuberculosis H37Rv and its heterologous expression and purification
Journal name Journal of Bacteriology   Check publisher's open access policy
ISSN 0021-9193
Publication date 2007-09-01
Sub-type Article (original research)
DOI 10.1128/JB.00425-07
Open Access Status File (Publisher version)
Volume 189
Issue 17
Start page 6246
End page 6252
Total pages 7
Place of publication Washington, DC, United States
Publisher American Society for Microbiology
Language eng
Abstract The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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