Lüttgen H, Rohdich F, Herz S, Wungsintaweekul J, Hecht S, Schuhr C A, Fellermeier M, Sagner S, Zenk M H, Bacher A, Eisenreich W
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1062-7. doi: 10.1073/pnas.97.3.1062.
A comparative analysis of all published complete genomes indicated that the putative orthologs of the unannotated ychB gene of Escherichia coli follow the distribution of the dxs, dxr, and ygbP genes, which have been shown to specify enzymes of the deoxyxylulose phosphate pathway of terpenoid biosynthesis, thus suggesting that the hypothetical YchB protein also is involved in that pathway. To test this hypothesis, the E. coli ychB gene was expressed in a homologous host. The recombinant protein was purified to homogeneity and was shown to phosphorylate 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP-dependent reaction. The reaction product was identified as 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by NMR experiments with various (13)C-labeled substrate samples. A (14)C-labeled specimen of this compound was converted efficiently into carotenoids by isolated chromoplasts of Capsicum annuum. The sequence of E. coli YchB protein is similar to that of the protein predicted by the tomato cDNA pTOM41 (30% identity), which had been implicated in the conversion of chloroplasts to chromoplasts.
对所有已发表的完整基因组进行的比较分析表明，大肠杆菌未注释的ychB基因的假定直系同源基因遵循dxs、dxr和ygbP基因的分布模式，这些基因已被证明参与萜类生物合成的脱氧木酮糖磷酸途径中酶的特异性表达，因此表明假定的YchB蛋白也参与该途径。为了验证这一假设，在同源宿主中表达了大肠杆菌ychB基因。重组蛋白被纯化至同质，并显示在依赖ATP的反应中使4-二磷酸胞苷-2C-甲基-D-赤藓糖醇磷酸化。通过使用各种（13）C标记底物样品的NMR实验，反应产物被鉴定为4-二磷酸胞苷-2C-甲基-D-赤藓糖醇2-磷酸。该化合物的（14）C标记标本被辣椒分离的有色体有效地转化为类胡萝卜素。大肠杆菌YchB蛋白的序列与番茄cDNA pTOM41预测的蛋白序列相似（同一性为30%），该蛋白与叶绿体向有色体的转化有关。
