E nol p yruvyl s hikimate p hosphate s ynthase

Abstract

lhe shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays 1. 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. lhe last step resulted in two well-separated adivities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial adivity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, adivation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. lwo EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. lhis 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 latelogarithmic phase. lhe 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. The sixth enzyme of the shikimate pathway leading to aromatic amino acid biosynthesis, EPSP synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19), cat