Dihydrodipicolinate synthase ofnicotiana sylvestris,a chloroplast-localized enzyme of the lysine pathway |
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Authors: | Marc Ghislain Valerie Frankard Michel Jacobs |
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Institution: | (1) Laboratory of Plant Genetics, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 St-Genesius Rode, Belgium |
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Abstract: | The first enzyme of the lysine-biosynthesis pathway, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has been purified and
characterized inNicotiana sylvestris Speggazini et Comes. A purification scheme was developed for the native DHDPS that subsequently led to the purification to
homogeneity of its subunits using two-dimensional gel electrophoresis. Subsequent elution of the purified polypeptide has
opened the way for the production of rabbit polyclonal anti-DHDPS sera. The molecular weight of the enzyme was determined
to be 164000 daltons (Da) by an electrophoretic method. By labeling with 14C]pyruvate, the enzyme was shown to be composed of four identical subunits of 38500 Da. Pyruvate acts as a stabilizing agent
and contributes to the preservation of the tetrameric structure of the enzyme. The enzyme ofN. sylvestris is strongly inhibited by lysine with anI
0.5
of 15 μM; S-(2-aminoethyl)L-cysteine and γ-hydroxylysine, two lysine analogs, were found to be only weak inhibitors. An analog of pyruvate, 2-oxobutyrate,
competitively inhibited the enzyme and was found to act at the level of the pyruvate-binding site. Dihydrodipicolinate synthase
was localized in the chloroplast and identified as a soluble stromal enzyme by enzymatic and immunological methods. Its properties
are compared with those known for other plant and bacterial DHDPS enzymes. |
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Keywords: | Dihydrodipicolinate synthase (purification subcellular localization) Lysine biosynthesis Nicotiana (lysine biosynthesis) |
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