Abstract
Coding regions of a cDNA for precursor and mature chorismate synthase (CS), a plastidic enzyme, from Corydalis sempervirens were expressed in Escherichia coli as translational fusions to glutathione-S-transferase. Fusion proteins were purified, and precursor and mature forms of CS were then released by proteolytic cleavage with factor Xa. Although mature CS was enzymatically active after release, activity could be detected neither for the precursor CS nor for corresponding glutathione-S-transferase fusion proteins. In contrast, two other shikimate pathway enzymes (shikimate kinase and 5-enol-pyruvylshikimate-3-phosphate synthase) have previously been shown to be as enzymatically active as their respective higher molecular weight precursors. By expression of unfused, mature CS from C. sempervirens in E. coli, it was possible to obtain large quantities of enzymatically active CS protein compared to yields from plant cell cultures. Expression levels in E. coli approached 1% of total soluble protein. No differences were found between authentic CS isolated from cell cultures and CS expressed in and purified from E. coli, which made possible a more detailed biochemical characterization of CS. Quaternary structure analysis of the purified mature CS indicated that the enzyme exists as a dimer, in contrast to the active tetrameric structures determined for E. coli and Neurospora crassa enzymes.
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