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The Two Chorismate Mutases from both Mycobacterium tuberculosis and Mycobacterium smegmatis: Biochemical Analysis and Limited Regulation of Promoter Activity by Aromatic Amino Acids

Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, Porto Alegre, RS 90619-900, Brazil,¹ Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom,² Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Porto Alegre, RS 91501-970, Brazil³

Received 16 August 2007/ Accepted 17 October 2007

Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as "conserved hypothetical proteins" in the M. tuberculosis genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the M. tuberculosis enzymes are also present in Mycobacterium smegmatis, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three M. tuberculosis loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using M. smegmatis as a surrogate host. Our results indicate that the aroQ (Rv0948c), *aroQ (Rv1885c), and fbpB (Rv1886c) genes from M. tuberculosis are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.

Published ahead of print on 26 October 2007.