5-ENOL-PYRUVYL-SHIKIMATE-3-PHOSPHATE SYNTHASE FROM ZEA-MAYS CULTURED-CELLS

The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPS P) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.1 9) was purified from cultured maize (Zea mays L. var Black Mexican Swe et) cells. Homogeneous enzyme preparations were obtained by a four-ste p procedure using ammonium sulfate fractionation, anion- and cation-ex change chromatography, and substrate elution from a cellulose phosphat e column. The last step resulted in two well-separated activities of a bout the same molecular weight. A 2000- to 3000-fold purification, wit h an overall recovery of one-fourth of the initial activity, was achie ved. Both EPSP synthase isoforms were characterized with respect to st ructural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinitie s for the two substrates. A more pronounced difference was found betwe en their thermal inactivation rates. Two EPSP synthase isoforms were a lso elucidated in crude homogenates by anion-exchange fast protein liq uid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throu ghout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified pl astid preparations demonstrated a plastidial localization of both prot eins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on d efense-related aromatic biosynthesis in higher plants, are discussed.