THE FIRST ENZYME OF THE SHIKIMATE PATHWAY FROM POTATO (SOLANUM TUBEROSUM L. CV. SUPERIOR) (DAHP SYNTHASE)

JOSE EDUARDO BRASIL PEREIRA PINTO, Purdue University

Abstract

The enzyme that catalyzes the first step of the shikimate pathway, the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), was demonstrated in potato (Solanum tuberosum L. cv. Superior) tuber, leaf, root, stem, stolon, in potato cells grown in suspension culture, and in several other plants. The enzyme from potato tuber was purified to electrophoretic homogeneity. The enzyme is a dimeric protein with subunits of 54,000 daltons. The enzyme activity has a narrow pH optimum around pH 7.0. The enzyme is activated by Mn('2+) and tryptophan. The enzyme is hysteretic; tryptophan activation eliminates the hysteresis. Rabbit antibodies were raised against the pure enzyme. The antibodies cross-reacted with DAHP synthase from all parts of the potato plant, as shown by Ouchterlony double diffusion and immunoprecipitation from solution. The antibodies were monospecific as judged by immunodiffusion and immunoelectrophoresis. Antigenic similarities between DAHP synthases from different C(,3) and C(,4) plants were shown by immunoprecipitation from solution. The amount of DAHP synthase polypeptide in various parts of the potato plant was quantitated by rocket immunoelectrophoresis. The largest amount and the highest overall activity of the enzyme was found in the young leaf. Growing stems had the highest specific enzyme activity. The ratios of specific enzyme activity to amount of DAHP synthase polypeptide were not the same for the enzyme from different plant parts or within the potato root during plant development. I interpreted these data to indicate modification of enzyme activity, because I found no evidence for differentially expressed isoenzymes. Subsequently, I demonstrated that the enzyme from Solanum tuberosum cells grown in suspension culture is activated by posttranslational modification, when the cells are challenged with sublethal doses of N-(phosphonomethyl)glycine, the herbicide known as glyphosate which inhibits the penultimate enzyme of the shikimate pathway. In vitro, DAHP synthase from cells of Solanum tuberosum grown in suspension culture is not affected by glyphosate. The in vivo activation of the enzyme demonstrates convincingly that potato DAHP synthase is a regulatory enzyme. Protoplasts, prepared from Solanum tuberosum cells grown in culture, were used for subcellular fractionation of cell organelles. The DAHP synthase was located exclusively in the plastidic fraction of the cell.

Degree

Ph.D.

Subject Area

Botany

COinS