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J. Bacteriol. doi:10.1128/JB.00425-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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, RS 90619-900, Brazil; Laboratório de Biologia Estrutural e Zooquímica, Centro de Estudos de Insetos Sociais, Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro - SP 13506-900, Brazil; Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 91501-970, Brazil

^* To whom correspondence should be addressed. Email: luiz.basso{at}pucrs.br. diogenes{at}pucrs.br.

	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 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 plant, fungi, bacteria and apicomplexan parasites. The aroB-encoded dehydroquinate synthase is the second enzyme of this pathway and it catalyzes the cyclisation of 3-deoxy-D-arabino-heptulosonate 7-phosphate in 3-dehydroquinate. Here, we describe the PCR amplification and cloning of aroB gene, and overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe whereas the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The mutant E. coli AB2847, was used to demonstrate that the plasmid construction was enable to repair the mutants, allowing them to grow in minimal medium devoided of aromatic compounds supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes thereby showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for rational design of antimycobacterial agents.