Laboratory of Plant Genetics, Vrije Universiteit Brussel, Paardenstraat 65, B-1640, St-Genesius Rode, Belgium.
Planta. 1990 Mar;180(4):480-6. doi: 10.1007/BF02411444.
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 [(14)C]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.
赖氨酸生物合成途径的第一个酶，二氢二吡啶羧酸合酶（DHDPS；EC 4.2.1.52）已在Nicotiana sylvestris Speggazini et Comes 中被纯化和表征。开发了一种用于天然 DHDPS 的纯化方案，随后使用二维凝胶电泳将其亚基纯化至均一性。随后对纯化的多肽进行洗脱，为生产兔多克隆抗 DHDPS 血清开辟了道路。通过电泳方法确定该酶的分子量为 164000 道尔顿（Da）。通过用 [(14)C]丙酮酸标记，证明该酶由四个相同的 38500 Da 亚基组成。丙酮酸作为稳定剂，有助于保持酶的四聚体结构。来自N. sylvestris 的酶被赖氨酸强烈抑制，I0.5 为 15 μM；发现 S-(2-氨基乙基)L-半胱氨酸和γ-羟基赖氨酸这两种赖氨酸类似物只是弱抑制剂。丙酮酸的类似物 2-氧代丁酸竞争性抑制该酶，被发现作用于丙酮酸结合位点。DHDPS 定位于叶绿体中，并通过酶学和免疫学方法鉴定为可溶性基质酶。其性质与其他植物和细菌 DHDPS 酶的性质进行了比较。
