Cytidine 5'-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol

Abstract

2-C-methylerythritol 4-phosphate has been established recently as an intermediate of the deoxyxylulose phosphate pathway used for biosynthesis of terpenoids in plants and in many microorganisms. We show that an enzyme isolated from cell extract of Escherichia coli converts 2-C-methylerythritol 4-phosphate into 4-diphosphocytidyl-2-C-methylerythritol by reaction with CTP. The enzyme is specified by the hitherto unannotated ORF ygbP of E. coli. The cognate protein was obtained in pure form from a recombinant hyperexpression strain of E. coli harboring a plasmid with the ygbP gene under the control of a T5 promoter and lac operator. By using the recombinant enzyme, 4-diphosphocytidyl-[2-(14)C]2-C-methylerythritol was prepared from [2-(14)C]2-C-methylerythritol 4-phosphate. The radiolabeled 4-diphosphocytidyl-2-C-methylerythritol was shown to be efficiently incorporated into carotenoids by isolated chromoplasts of Capsicum annuum. The E. coli ygbP gene appears to be part of a small operon also comprising the unannotated ygbB gene. Genes with similarity to ygbP and ygbB are present in the genomes of many microorganisms, and their occurrence appears to be correlated with that of the deoxyxylulose pathway of terpenoid biosynthesis. Moreover, several microorganisms have genes specifying putative fusion proteins with ygbP and ygbB domains, suggesting that both the YgbP protein and the YgbB protein are involved in the deoxyxylulose pathway. A gene from Arabidopsis thaliana with similarity to ygbP carries a putative plastid import sequence, which is well in line with the assumed localization of the deoxyxylulose pathway in the plastid compartment of plants.

Figure 1

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis.

Figure 2

Reaction catalyzed by YgbP protein (4-diphosphocytidyl-2-C-methylerythritol synthase).

Figure 3

Deduced amino acid sequences of ygbP and ygbB genes. Residues conserved in more than 50% of the sequences are shown in inverse contrast. A–L, putative YgbP proteins; M and N, bifunctional YgbP/YgbB proteins; O-X, YgbB proteins. A, E. coli; B, H. influenzae; C, B. subtilis; D, M. tuberculosis; E, Synechocystis sp. PC6803; F, Aquifex aeolicus; G, Chlamydia trachomatis; H, Chlamydia pneumonia; I, Thermotoga maritima; J, P. horikoshii; K, P. falciparum(fragment); L, A. thaliana; M, H. pylori; N, T. pallidum; O, E. coli; P, H. influenzae; Q, B. subtilis; R, M. tuberculosis; S, Synechocystis sp. PC6803; T, A. aeolicus; U, C. trachomatis; V, C. pneumonia; W, T. maritima; X, P. falciparum.

Figure 4

SDS/PAGE. Lane 1, molecular mass markers; lane 2, crude cell extract of the recombinant E. coli strain expressing YgbP protein; lane 3, YgbP protein after Sepharose Q chromatography; lane 4, YgbP protein after Red Sepharose chromatography; lane 5, YgbP protein after Source 15Q chromatography.