PLANT PHYSIOLOGY , Vol 108, Issue 3 1127-1132, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Only the Mature Form of the Plastidic Chorismate Synthase Is Enzymatically Active
J. M. Henstrand, J. Schmid and N. Amrhein
Institute of Plant Sciences, Swiss Federal Institute of Technology, Universitatstrasse 2, CH-8092 Zurich, Switzerland
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.
