This Article Full Text Full Text (PDF) Other Versions of this Article: JB.00425-07v1 189/17/6246    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by de Mendonça, J. D. Articles by Santos, D. S. Search for Related Content PubMed PubMed Citation Articles by de Mendonça, J. D. Articles by Santos, D. S.

Functional Characterization by Genetic Complementation of aroB-Encoded Dehydroquinate Synthase from Mycobacterium tuberculosis H37Rv and Its Heterologous Expression and Purification

Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul 90619-900,¹ Laboratório de Biologia Estrutural e Zooquímica, Centro de Estudos de Insetos Sociais, Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, São Paulo 13506-900,² Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul 91501-970, Brazil³

Received 22 March 2007/ Accepted 11 June 2007

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.

Published ahead of print on 22 June 2007.