Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana

Abstract

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 micromol small middle dotmin(-1) mg(-1). The Michaelis constants for 4-diphosphocytidyl-2C-methyl-d-erythritol and CTP were 500 microM and 114 microM, respectively.

Figure 1

The nonmevalonate pathway of isoprenoid biosynthesis.

Figure 2

Intron/exon topology of the ispD gene of A. thaliana.

Figure 3

ORF and predicted amino acid sequence of the 4-diphosphocytidyl-2C-methyl-d-erythritol synthase gene of A. thaliana. The amino acid sequence of the pseudomaturated catalytic domain is shaded.

Figure 4

SDS/PAGE. Lane A, molecular weight markers; B, cell extract of E. coli XL1-Blue; C, cell extract of recombinant E. coli expressing the full-length 4-diphosphocytidyl-2C-methyl-d-erythritol synthase of A. thaliana; D, cell extract of recombinant E. coli expressing the catalytic domain of 4-diphosphocytidyl-2C-methyl-d-erythritol synthase of A. thaliana; E, catalytic domain of 4-diphosphocytidyl-2C-methyl-d-erythritol synthase after Source 15 Q chromatography; catalytic domain of 4-diphosphocytidyl-2C-methyl-d-erythritol synthase after Superdex G 200 gel filtration.

Figure 5

Two-dimensional heteronuclear multiple quantum correlation spectrum of [1,3,4-¹³C]4-diphosphocytidyl-2C-methyl-d-erythritol (¹H and ¹³C NMR signal assignments from ref. 14).

Figure 6

Double reciprocal Lineweaver–Burk plot of 4-diphosphocytidyl-2C-methyl-d-erythritol synthase from A. thaliana. (A) Various amounts of CTP; (B) various amounts of 2C-methyl-d-erythritol 4-phosphate.

Figure 7

Boundary sedimentation of the recombinant catalytic domain of 4-diphosphocytidyl-2C-methyl-d-erythritol synthase from A. thaliana at 60,000 rpm and 20°C. (A) Low-salt buffer (50 mM Tris⋅hydrochloride, pH 8.0; (B) high-salt buffer (20 mM potassium citrate/200 mM potassium phosphate, pH 6.6). Scans were obtained at intervals of 5 min. Absorbance was monitored at 280 nm.

Figure 8

Alignment of deduced amino acid residues of 4-diphosphocytidyl-2C-methyl-d-erythritol synthases. Lane A, A. thaliana [AF230737]; Lane B, E. coli [AF230736]. Identical residues are shown in inverse contrast; similar residues are shaded.