Shikimate Kinase; Searching for Potential Novel Anti-Tubercular Agents

Tuberculosis is a respiratory infection with over 10 million reported cases each year. This infection rate is responsible for over two million deaths annually, second only to HIV in fatalities among infectious diseases annually. Studies in the last decade have shown that tuberculosis rates have gradually increased. This observation suggests that there is an alarming increase in the prevalence of drug resistant strains of tuberculosis, thus the need for the discovery of novel antitubercular agents. The shikimate pathway is a seven step metabolic route that produces aromatic amino acids and other cellular metabolites. The target enzyme in this project, Mycobacterium tuberculosis Shikimate Kinase (MtSK), catalyzes the 5th step of this pathway. MtSK converts shikimate to shikimate 3 phosphate. The overall goal of this project is to express and characterize MtSK to screen for potential antitubercular agents. Initial methods included a bacterial transformation of XL1 blue competent E.coli cells. This preliminary transformation was performed using a pET-21b plasmid with an aroK gene inserted into the multiple cloning site. Successfully transformed XL1 blue cells were cloned and a second transformation of BL21 DE3 competent cells was performed. Initial small scale expression showed the presence of a band around 20 kDa. The theoretical mass of the enzyme is 19.6 kDa which suggests MtSK was successfully expressed. Expression analysis for large scale supported data from small scale as a band of 20 kDa was present. The purity and molecular weight of MtSK were confirmed by nickel affinity chromatography as well as ESI-LC-MS. ATP-dependent kinetic parameters determined via a spectrophotometric coupled assay showed a kcat of 50 ± 6 mM, KM of 0.2 ± 0.04 mM. shikimate dependent kinetic parameters showed a kcat of 55 ± 2 mM, KM of 1.8 ± 0.1 mM. Quenching intrinsic tyrosine fluorescence showed binding affinities similar to ATP and shikimate-dependent parameters. The marine compound, avarone showed substantial quenching of intrinsic fluorescence. The inhibitory mechanism of avarone on MtSK is currently under investigation. © FASEB.