Overexpression, Purification, and Use of Phosphoenol Pyruvate Synthetase in the Synthesis of PEP Analogues

TheEscherichia colienzyme phosphoenol pyruvate synthetase has been overexpressed and purified in a single chromatographic step. The enzyme catalyzes the synthesis of phosphoenol pyruvate (PEP), from adenosine triphosphate and pyruvate, and has enabled the synthesis of uniformly labeled [1,2,3-13C3]PEP, which is a key molecule in structural and mechanistic studies of enolpyruvyl transferases. Fluoropyruvate was also used as substrate for the enzyme and gave only (Z)-phoephoenol-3-fluoropyruvate, albeit at a slower rate.