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.