The shikimate pathway constitutes the initial stage of the biosynthesis of aromatic amino acids. This pathway is present in several organisms as plants, fungi, bacteria and apicomplexan parasites. Despite that fact, it was reported to be essential in plants and microorganisms such as Mycobaterium tuberculosis. The increase of multi-drug resistant M. tuberculosis strains makes the enzymes of this pathway important targets for the development of new drugs. One of the most well characterized enzymatic inhibitor of shikimate pathway is the glyphosate, which inhibits the enolpyruvylshikimate-3-phosphate synthase (EPSPS) (EC 2.5.1.19). The endogenous substrate of this enzyme, the shikimate-3-phosphate (S3P), is needed for structural and kinetic characterization of EPSPS, but it is not available to purchase. This account summarizes a new synthesis of S3P based on an enzymatic method using M. tuberculosis shikimate kinase (MtSK), which catalyzes the transfer of the phosphate from ATP to the 3-hydroxyl group of shikimic acid.
          We have previously cloned, expressed and purified the MtSK (Oliveira et al, 2003). A solution of 120 mM shikimic acid, 1,2 mM ATP, 15 mM PEP, 2,5 U MtSK and 2,5 U PK were incubated at 25°C for 30 minutes in 100 mM Tris 100mM KCl 5 mM MgCl₂ pH 7,6 buffer. All reagents, except the MtSK, were purchased from Sigma. The reaction mixture was ultrafiltrated in a Centricon membrane (10 KDa). The filtrated fraction was subjected to anionic exchange chromatography in a MonoQ 10/10 column (GE-Healthcare) under the follow gradient: 0,2 M to 0,8 M NH₄HCO₃ pH. 9.0 buffer. The fractions were monitored by the enzymatic activity of EPSPS. The purified product was precipitated with 1 M barium acetate, washed tree times with ethanol and dried on reduced pressure. The characterization of the barium salt of S3P was made by spectroscopy methods (UV – 190 nm to 400 nm - and MS), which confirmed the identity of the product and its purity of 95%.
          The EPSPS from M. tuberculosis was cloned, expressed and purified. The pure barium salt of S3P will be used in the structural and kinetic characterization of EPSPS, which will provide important data to the development of enzymatic inhibitors.