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

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.

Molecular cloning, expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor

“…Typically, production hosts contain a prophage (DE3) encoding the highly processive T7 RNA polymerase under control of the IPTG-inducible lacUV5 promoter that would ensure tight control of recombinant gene basal expression [31]. In agreement with the results presented here, high levels of protein expression in the absence of inducer have been shown to occur in the pET system [32][33][34][35][36]. It has been proposed that leaky protein expression is a property of lac-controlled system when cells approach stationary phase in complex medium and that cyclic AMP, acetate, and low pH are required to achieve high-level expression in the absence of IPTG induction, which may be part of a general cellular response to nutrition limitation [37].…”

New tuberculosis drug development: targeting the shikimate pathway

“…[90] ; fi rst mechanism-based inhibitors synthesized [73] 2. DHQ synthase aroB (Rv2538c) 38.1 kDa [86] 1089 bp 362 amino acids Essential for ring-containing compound synthesis MT DHQ synthase crystal structure complete [109] , mechanism studies underway [43] ; carbocyclic phosphonate (DAHP analogue) inhibition studies [110] for further catalysis understanding needed 3. DHQ dehydratase aroD (Rv2537c) 15.8 kDa [43] 444 bp 147 amino acids Introduction of fi rst double bond to aromatic ring MT crystal structure complete [111] ; inhibitory action observed in series of previously known inhibitors [93] 4.…”

“…All the enzymes of the M. tuberculosis shikimate pathway have been characterized to some degree [82][83][84][85][86][87][88] . However, the number of studies investigating M. tuberculosis crystal structures and mechanisms of action vary from enzyme to enzyme.…”

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

“…Since this pathway is absent in human host and is essential for mycobacteria, its seven enzymes are attractive targets for the development of antitubercular agents [8,9]. Since the catalytic chemistry of enzymes is the key to designing potent inhibitors and making them special class of drug target [10] efforts have been made to characterize at the molecular and biochemical levels many enzymes from this pathway in M. tuberculosis, such as 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase [11], dehydroquinate synthase [12], shikimate kinase [13], 5-enolpyruvylshikimate-3-phosphate synthase [14], and chorismate synthase [15,16].…”