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

Abstract

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. Tuberculosis (TB), a serious infectious disease caused byMycobacterium tuberculosis, still remains a public health problem in the world. It has been estimated that one-third of the world's population is latently infected with M. tuberculosis and that almost 9 million new cases and 2 million deaths occur each year (19). The low efficacy of the currently available TB vaccine (Mycobacterium bovis BCG), the emergence of multidrug-resistant strains of M. tuberculosis, and the global spread of human immunodeficiency virus (which increases the risk of TB development) are among the factors underlying the resurgence of TB (24). New drugs and second-generation vaccines are urgently required to control TB, but the complex biology of M. tuberculosis has hindered the development of novel therapeutic tools.M. tuberculosis is a sophisticated intracellular pathogen that can persist for months or years within the human host, and different mechanisms related to its entry, survival, and replication in macrophages are involved in the infection process (51, 56). The bacilli are notably able to interfere with normal macrophage functions, such as antigen presentation and phagosome maturation, residing in a specialized, nonacidified compartment (44). Of particular interest is the fact that important nutrients are restricted or unavailable inside macrophages, so that M. tuberculosis cells are probably subjected to long-term starvation during the early and latent stages of infection (6,36,50). Since many anabolic and catabolic pathways should be specifical...

Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum

“…RHA1, and nfa49960 (57 %) of Nocardia farcinica IFM 10152. CM0819 also showed sequence similarity to functionally identified CMs, the highest similarity being to the recently identified cytoplasmic MtbCM (encoded by Rv0948c, identity 57 %) (Schneider et al, 2008), and to previously identified EcCM from E. coli (27 %), MjCM from M. jannaschii (10.7 %), ScCM from S. cerevisiae (9.4 %), HpCM from H. polymorpha (7.9 %), and AtCM3 from A. thaliana (7.4 %). An alignment of the deduced CMs and the structurally characterized E. coli CM revealed a range of conserved amino acid residues (data not shown).…”

“…Chorismate mutases (CMs, EC 5.4.99.5) are of interest for bioindustry and medicine, because the enzyme is the target to improve production of aromatic amino acids such as Lphenylalanine (Phe) and L-tyrosine (Tyr) (Chavez-Bejar et al, 2008;Yakandawala et al, 2008) as well as being a putative new drug target against deadly diseases such as tuberculosis (Qamra et al, 2006;Schneider et al, 2008). CMs catalyse the conversion of chorismate to prephenate, a reaction also known as the Claisen rearrangement, and the only naturally occurring Claisen reaction in the primary metabolism of living organisms (Krappmann et al, 1999;Sugimoto & Shiio, 1980a).…”

“…The AroQ class CMs are further divided into cytoplasmic and monofunctional CMs that are not subject to allosteric regulation [e.g. Rv0948c from Mycobacterium tuberculosis (Schneider et al, 2008)], periplasmic/secreted and monofunctional CMs [e.g. Rv1885c from M. tuberculosis (Prakash et al, 2005)], and multiple functional fusion CMs [e.g.…”

The Rv1712 Locus from Mycobacterium tuberculosis H37Rv Codes for a Functional CMP Kinase That Preferentially Phosphorylates dCMP

“…These structural differences could be exploited in the development of novel inhibitors targeted more specifically toward M. tuberculosis and other pathogenic mycobacteria. The results presented here provide a solid foundation to carry out genetic studies (16) to demonstrate the role, if any, of cmk in M. tuberculosis survival in the human host. …”

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane ofGluconobacter oxydansIFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

“…A similar strategy can be applied to control parasites as anti-malarial agents. [5][6][7][8][9] In this study, to facilitate the production of metabolic intermediates of pharmaceutical significance in the SKA pathway, immobilization of the membrane fraction of G. oxydans IFO 3244, having the ability to catalyze oxidative fermentation from quinate to DSA, was examined. Immobilization of the cytoplasmic asymmetric reduction system composed of SKDH and GDH was also examined to identify the best carrier for SKA formation from DSA.…”