Indexed on: 14 Feb '12Published on: 14 Feb '12Published in: Protein Expression and Purification
3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS), (EC 18.104.22.168) catalyzes the first step of the shikimate pathway, the route for the biosynthesis of aromatic compounds in plants and microbes. In Actinosynnema pretiosum, the aroF gene (GenBank: AF056968.1) encodes DAHPS to condensate phosphoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to generate DAHP. In this study, a recombinant pET28a-aroF plasmid was constructed and A. pretiosum DAHPS was successfully expressed in soluble form by co-expression with chaperonins GroEL/GroES in Escherichia coli. The purification and kinetic characterization of the expressed protein were then investigated. The DAHPS originated from A. pretiosum demonstrated a pronounced substrate inhibition by PEP but was not sensitive to E4P. The purified enzyme was completely inactivated by EDTA but potently activated by several bivalent metal ions, especially Mn(2+) and Co(2+).