REGULATION OF AROMATIC AMINO ACID BIOSYNTHESIS IN HIGHER PLANTS: THE PURIFICATION AND CHARACTERIZATION OF DAHP SYNTHASES FROM CARROTS AND SPINACH

JEFFREY FRANCIS DAVID DEAN, Purdue University

Abstract

Carrot root contains three forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the first enzyme of the shikimate pathway. The enzymes are activated by Mn('2+), tryptophan, and indole acetic acid (IAA), require reducing agents to maintain activity, and are irreversibly inhibited by ascorbate. One of the activities is purified to homogeneity by a method that improves on previously known procedures. The K(,m) values of the purified enzyme for PEP and E4P are 0.03 and 0.07 mM, respectively. High-titre antibodies raised against the purified enzyme crossreact with the other two forms, allowing identification by immunoblotting of all three partially purified proteins. The three proteins are very similar as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis and limited proteolysis. When carrot root is extracted by a different procedure, a fourth DAHP synthase is found that requires Co('2+) or Mg('2+) for activity, and is unresponsive to ascorbate. Mn('2+)-activated DAHP synthase specific enzyme activity changes during growth of carrot cells in suspension culture. The activity changes are reflected in variation of the relative amounts of the three enzyme forms. Lysates of carrot cell protoplasts were fractionated by centrifugation on sucrose gradients. Analysis of the fractions suggest that the Mn('2+)-activated DAHP synthase is localized in the plastids while the Co('2+)/Mg('2+)-activated enzyme is in the cytosol. Extracts of spinach leaves contain a Mn('2+)-activated and a Co('2+)/Mg('2+)-activated DAHP synthase. The Mn('2+)-activated DAHP synthase has a molecular weight of 53,000, and is localized in the spinach chloroplasts. The enzyme is activated by tryptophan, and IAA, but inhibited by ascorbate. The enzyme crossreacts with antibodies raised against carrot DAHP synthase. Extracts of carrot, spinach leaf, and mung bean seedlings contain 3-deoxy-D-manno-octulosonate 8-phosphate (DMOP) synthase. The enzyme catalyzes the condensation of PEP and arabinose 5-phosphate to DMOP and orthophosphate, an activity previously described in bacteria. However, this thesis presents the first description of DMOP synthase from any higher plant.

Degree

Ph.D.

Subject Area

Biochemistry

COinS