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.

Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate to 2C-methyl- d -erythritol 2,4-cyclodiphosphate

“…These observations suggested that YgbB protein is involved in the deoxyxylulose phosphate pathway of terpenoid biosynthesis. This hypothesis was supported further by the fact that putative orthologs of dxr, dxs, ygbP, ychB, and ygbB are found in all completely sequenced genomes of microorganisms using the nonmevalonate pathway and are absent in all completely sequenced genomes of microorganisms using the mevalonate pathway with the exception of Pyrococcus horikoshii genome, which contains a putative ortholog of ygbP (15,16). The ygbB gene of E. coli was expressed in a recombinant E. coli host harboring the plasmid pQEygbB, which directs the synthesis of a modified YgbB protein with six consecutive histidine residues at the N terminus.…”

“…31 P 13 C coupling of one phosphorous atom to the 2-methyl carbon and the absence of 31 P 1 H coupling for the 31 P NMR signal at Ϫ11.66 ppm (Figs. 3 and 4), in conjunction with the 13 C NMR chemical shift for C-2 of the 2C-methyl-D-erythritol moiety (83.9 ppm) as compared with the respective chemical shift for C-2 of 4-diphosphocytidyl-2C-methyl-D-erythritol (73.8 ppm) (15) clearly indicated the connection of the diphosphate motif to C-2 (Table 3). These data established the structure as 2C-methyl-Derythritol 2,4-cyclodiphosphate (8, Fig.…”

“…The ygbP gene specifying 4-diphosphocytidyl-2C-methyl-Derythritol synthase is tightly linked to the unannotated reading frame ygbB on the E. coli chromosome (15,22), and the two genes are likely to be cotranscribed. Putative orthologs of ygbP and ygbB also were found in the database in adjacent positions on the chromosomes of 21 other eubacteria (Table 2), and 6 eubacteria were found to contain genes assumed to specify bifunctional proteins with an N-terminal ygbP domain and a C-terminal ygbB domain (Table 2).…”

“…1-Deoxy-D-xylulose 5-phosphate was shown to be converted into the branched chain polyol 2C-methyl-D-erythritol 4-phosphate (4) by reductoisomerases specified by dxr genes of E. coli, Mentha piperita, Arabidopsis thaliana, and Plasmodium falciparum (17)(18)(19)(20). More recently, we found that 2C-methyl-D-erythritol 4-phosphate can be converted into 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (6) via 4-diphosphocytidyl-2C-methyl-D-erythritol by the sequential action of enzymes specified by the ygbP and ychB genes of E. coli (15,16). This paper shows that YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into the cyclic 2,4-diphosphate of 2C-methyl-D-erythritol.…”

Biosynthesis of Isoprenoids via Mevalonate in Archaea: The Lost Pathway

“…DXP is converted into 2-C-methylerythritol 4-phosphate by DXP reductoisomerase, an enzyme that could have been recruited by merging a unique C-terminal half with a glycinerich N-terminal domain related to the dinucleotidebinding site of many oxidoreductases (Takahashi et al 1998). 2-C-methylerythritol 4-phosphate in E. coli is converted into 4-diphosphocytidyl-2-C-methylerythritol by the YgbP protein (Rohdich et al 1999), a nucleotidyltransferase of the large and widespread GlmU family (A.R. Mushegian, unpubl.…”