Purification and characterization of chorismate synthase from Euglena gracilis. Comparison with chorismate synthases of plant and microbial origin

• Published in: Plant physiology (Bethesda) Vol. 97; no. 4; pp. 1271 - 1279
• Main Authors: Schaller, A. (Swiss Federal Institute of Technology, Zurich, Switzerland), Afferden, M. van, Windhofer, V, Bulow, S, Abel, G, Schmid, J, Amrhein, N
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.12.1991
• Subjects: ACIDE ORGANIQUE; ACIDOS ORGANICOS; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Antiserum; Bacteria; Biological and medical sciences; characterization; chemical kinetics; chorismate synthase; Chromatography; comparative studies; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Cyclic amino acids; enzymatic activity; Enzyme activity; Enzymes; Euglena; Euglena gracilis; Freshwater; Fundamental and applied biological sciences. Psychology; Fungi; Gels; LIGASAS; LIGASE; MASTIGOPHORA; Metabolism; METABOLISME; METABOLISMO; Phosphates; Plant physiology and development; PURIFICACION; PURIFICATION; Shikimate pathway; VIA BIOQUIMICA DEL METABOLISMO; VOIE BIOCHIMIQUE DU METABOLISME; Purification; Enzyme; Algae; Pyrrophycophyta; Fumariaceae; Immunological method; Fungi; Characterization; Flavin; Enzymatic activity; Dicotyledones; Euglena gracilis; Angiospermae; Bacteria; Ascomycetes; Spermatophyta; Neurospora crassa; Chorismate synthase; Kinetics; Comparative study; Thallophyta
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.97.4.1271

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