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 mu mol.min(-1) mg(-1). The Michaelis constants for 4-diphosphocytidyl-2C-methyl-D-erythritol and CTP were 500 mu M and 114 mu M, respectively.