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Metabolism and Enzymology

Purification and Characterization of Chorismate Synthase from Euglena gracilis ¹

Comparison with Chorismate Synthases of Plant and Microbial Origin

Institute of Plant Sciences, Swiss Federal Institute of Technology, Zürich, Switzerland, Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Federal Republic of Germany

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.

³ Present address: Institut für physiologische Chemie, Ruhr Universität Bochum, FRG.

⁴ Present address: PALL Biomedizin GmbH, Dreieich, FRG.

¹ Supported at Ruhr-Universität Bochum by a grant from the Deutsche Forschungsgemeinschaft and at the Swiss Federal Institute of Technology by ETHZ grant 07592/41-1030.5.

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