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 health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents. The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded 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-fold increase for EPSP synthase.
Oliveira
J S
JS
Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenido Bento Gonçalves 9500, Porto Alegre-RS 91501-970, Brazil.
Pinto
C A
CA
Basso
L A
LA
Santos
D S
DS
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D011994:Recombinant Proteins; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase