The shikimate pathway is comprised of seven enzymes and is utilized by plants, fungi, and bacteria to generate chorismate from the substrates phosphoenolpyruvate and erythrose-4-phosphate. In turn, chorismate is used as a precursor for biosynthesis of other important molecules such as aromatic amino acids, folate, and siderophores. Chorismate-utilizing enzymes are at the branchpoint between the shikimate pathway and these downstream pathways. In order to conduct kinetic isotope effect experiments on chorismate-utilizing enzymes, a sufficient quantity of isotopically labeled chorismate must be obtained. However, organic synthesis of labeled chorismate is laborious and low-yield. The goal of our research is to reconstitute the shikimate pathway <i>in vitro</i> by combining the enzymes, substrates, and cofactors in a one-pot reaction. Once the enzymes have been removed by filtration, the chorismate can then be isolated from the excess substrate and cofactors by HPLC. With deuterated phosphoenolpyruvate as a substrate, isotopically labeled chorismate can be generated using this approach. Development of an efficient one-pot reaction for chorismate will accelerate the kinetic and mechanistic studies for chorismate-utilizing enzymes.

Developing a method for one-pot synthesis of chorismate by <i>in vitro</i> reconstitution of the shikimate pathway

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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Developing a method for one-pot synthesis of chorismate by in vitro reconstitution of the shikimate pathway