Biosynthesis of terpenoids: 4-Diphosphocytidyl-2C-methyl-d-erythritol synthase of Arabidopsis thaliana

A hypothetical gene with similarity to the ispD gene of Escherichia coli was cloned from Arabidopsis thaliana cDNA. The ORF of 909 bp specifies a protein of 302 amino acid residues. The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78–202 bp. A fragment comprising amino acid residues 76–302 was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl-d-erythritol from 2C-methyl-d-erythritol 4-phosphate with a specific activity of 67 μmol⋅min(−1) mg(−1). The Michaelis constants for 4-diphosphocytidyl-2C-methyl-d-erythritol and CTP were 500 μM and 114 μM, respectively.

本研究从拟南芥（Arabidopsis thaliana）的互补脱氧核糖核酸（cDNA）中克隆得到一个与大肠杆菌（Escherichia coli）ispD基因具有序列相似性的推定基因。该基因的909碱基对（bp）开放阅读框（Open Reading Frame，ORF）可编码一条由302个氨基酸残基组成的蛋白质。其对应的染色体基因全长2071 bp，包含11个内含子，内含子长度范围为78~202 bp。研究人员将一段包含第76至302位氨基酸残基的蛋白片段在重组大肠杆菌菌株中进行了表达。该蛋白被纯化至均一状态，实验证实其可催化以2C-甲基-D-赤藓糖醇-4-磷酸为底物合成4-二磷酸胞苷-2C-甲基-D-赤藓糖醇的反应，比活性达67 μmol·min⁻¹·mg⁻¹。该酶对4-二磷酸胞苷-2C-甲基-D-赤藓糖醇与胞苷三磷酸（CTP）的米氏常数（Michaelis constant）分别为500 μM和114 μM。