ONLY THE MATURE FORM OF THE PLASTIDIC CHORISMATE SYNTHASE IS ENZYMATICALLY ACTIVE

Coding regions of a cDNA for precursor and mature chorismate synthase (CS), a plastidic enzyme, from Corydalis sempervirens were expressed i n Escherichia coli as translational fusions to glutathione-S-transfera se. Fusion proteins were purified, and precursor and mature forms of C S were then released by proteolytic cleavage with factor Xa. Although mature CS was enzymatically active after release, activity could be de tected neither for the precursor CS nor for corresponding glutathione- S-transferase fusion proteins. In contrast, two other shikimate pathwa y enzymes (shikimate kinase and 5-enol-pyruvylshikimate-3-phosphate sy nthase) have previously been shown to be as enzymatically active as th eir respective higher molecular weight precursors. By expression of un fused, mature CS from C. sempervirens in E. coli, it was possible to o btain large quantities of enzymatically active CS protein compared to yields from plant cell cultures. Expression levels in E. coli approach ed 1% of total soluble protein. No differences were found between auth entic CS isolated from cell cultures and CS expressed in and purified from E. coli, which made possible a more detailed biochemical characte rization of CS. Quaternary structure analysis of the purified mature C S indicated that the enzyme exists as a dimer, in contrast to the acti ve tetrameric structures determined for E. coli and Neurospora crassa enzymes.