Redox regulation of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

Abstract

The cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import. A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli. The resulting protein was purified to electrophoretic homogeneity. The enzyme requires Mn(2+) and reduced thioredoxin (TRX) for activity. Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 microM. The corresponding constant for TRX m is orders of magnitude higher. In the absence of TRX, dithiothreitol partially activates the enzyme. Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost. These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.

Figure 1

Purification of Arabidopsis DAHP synthase heterologously expressed in E. coli. SDS-PAGE on a 10% (w/v) polyacrylamide gel. Lane S, Five microliters of prestained M_r standards (Bio-Rad, Hercules, CA); lane 1, 10 μL of E. coli BL21(DE3)/pET23d/DHS1 crude extract; lane 2, 10 μL of the phosphocellulose pool; and lane 3, 100 μL of the Sephacryl 300 pool.

Figure 2

Metal ion activation of Arabidopsis DAHP synthase. The relative enzyme activity is plotted as a function of the metal ion concentration in the assay. ▪, MnCl₂; □, MgCl₂. Before assay, the purified enzyme was passed over a gel-filtration column equilibrated with a buffer containing neither MnCl₂ nor MgCl₂. The reaction was then started by adding the indicated concentrations of metal ions.

Figure 3

Activation of Arabidopsis DAHP synthase by reducing agents. The relative enzyme activity is plotted as a function of the concentration of the reducing agent in the assay. Before all assays, the purified enzyme was passed over a gel-filtration column equilibrated with a buffer that contained no reducing agent. A, The indicated concentrations of Spirulina sp. TRX were preincubated with 10 mm DTT and enzyme for 15 min at 25°C. The reactions were started by addition of substrates as described in “Materials and Methods.” B, Activation of the enzyme by DTT (squares) or β-mercaptoethanol (circles). C, Activation of the enzyme by recombinant spinach TRX f (squares) or TRX m (circles).

Figure 4

Alkylation of Arabidopsis DAHP synthase eliminates the need for reducing agent. A sample of the purified enzyme was divided into two equal portions. One portion was alkylated with iodoacetamide and the other as a control treated with water. Then both were passed over a Sephadex G25 column equilibrated with buffer C. Alkylated (squares) and nonalkylated (circles) enzyme preparations were kept at room temperature for the indicated times without DTT and then assayed in the presence (black symbols) or absence (white symbols) of DTT.

Figure 5

Biosynthesis of aromatic compounds in chloroplasts. Electrons of photosystem I (P₇₀₀*)-reduced Fd can either reduce NADP⁺ or TRX. Reduced TRX activates DAHP synthase, the first enzyme of the shikimate pathway.