PLANT PHYSIOLOGY , Vol 105, Issue 4 1107-1114, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties)
G. Forlani, B. Parisi and E. Nielsen
Department of Genetics and Microbiology, University of Pavia, I-27100 Pavia, Italy
The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP)
synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was
purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells.
Homogeneous enzyme preparations were obtained by a four-step procedure
using ammonium sulfate fractionation, anion- and cation-exchange
chromatography, and substrate elution from a cellulose phosphate column.
The last step resulted in two well-separated activities of about the same
molecular weight. A 2000- to 3000-fold purification, with an overall
recovery of one-fourth of the initial activity, was achieved. Both EPSP
synthase isoforms were characterized with respect to structural, kinetic,
and biochemical properties. Only slight differences are seen in molecular
mass, activation energy, and apparent affinities for the two substrates. A
more pronounced difference was found between their thermal inactivation
rates. Two EPSP synthase isoforms were also elucidated in crude homogenates
by anion-exchange fast protein liquid chromatography. This allowed us to
follow their expression during a culture growth cycle. One form was found
at substantial levels throughout, whereas the other increased in
exponentially growing cells and declined in late-logarithmic phase. The
analysis of highly purified plastid preparations demonstrated a plastidial
localization of both proteins. Possible functional roles for maize EPSP
synthase isozymes, with regard to the dual-pathway hypothesis and to the
recent findings on defense-related aromatic biosynthesis in higher plants,
are discussed.
