Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997. The reemergence of tuberculosis as a public heal th threat, the high susceptibility of HIV-infected persons, and the prolife ration of multidrug-resistant strains have created a need to develop new an timycobacterial agents. The existence of a shikimate pathway has been predi cted by the determination of the genome sequence of Mycobacterium tuberculo sis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-e nolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isoprop yl beta -D-thiogalactoside induction, and cells grown to stationary phase y ielded approximately 30% of target proteins to total soluble cell proteins. Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-f old increase for EPSP synthase. (C) 2001 Academic Press.