Cloning and expression of functional shikimate dehydrogenase (EC 1.1.1.25) from Mycobacterium tuberculosis H37Rv.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be one of the deadliest diseases in the world. TB resurged in the late 1980s and now kills more than 2 million people a year. Possible factors underlying the reemergence of TB are the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant (MDR) strains, patient noncompliance in completing the standard "short-course" therapy, and decline of health care systems. Thus, there is a need for the development of new antimycobacterial agents to treat MDR strains of M. tuberculosis, to provide for more effective treatment of latent tuberculosis infection, and to shorten the treatment course to improve patient compliance. The shikimate pathway is an attractive target for antimicrobial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent in mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of M. tuberculosis. The M. tuberculosis aroE-encoded shikimate dehydrogenase was PCR amplified, cloned, sequenced, and expressed in Escherichia coli BL21(DE3). Recombinant protein expression was achieved by a low-cost and simple protocol. Although cell lysis resulted in the formation of insoluble aggregates of the recombinant protein, soluble and functional M. tuberculosis shikimate dehydrogenase could be obtained by repeated cycles of freezing and thawing. Enzyme activity measurements demonstrated that there was approximately a 5-fold increase in specific activity for M. tuberculosis shikimate dehydrogenase. Moreover, the enzyme activity was linearly dependent upon the amount of recombinant protein added to the assay mixture, thus, confirming cloning and expression of functional mycobacterial shikimate.