Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl- d -erythritol

Abstract

A comparative analysis of all published complete genomes indicated that the putative orthologs of the unannotated ychB gene of Escherichia coli follow the distribution of the dxs, dxr, and ygbP genes, which have been shown to specify enzymes of the deoxyxylulose phosphate pathway of terpenoid biosynthesis, thus suggesting that the hypothetical YchB protein also is involved in that pathway. To test this hypothesis, the E. coli ychB gene was expressed in a homologous host. The recombinant protein was purified to homogeneity and was shown to phosphorylate 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP-dependent reaction. The reaction product was identified as 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by NMR experiments with various 13 C-labeled substrate samples. A 14 C-labeled specimen of this compound was converted efficiently into carotenoids by isolated chromoplasts of Capsicum annuum. The sequence of E. coli YchB protein is similar to that of the protein predicted by the tomato cDNA pTOM41 (30% identity), which had been implicated in the conversion of chloroplasts to chromoplasts.

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.…”

“…§1734 solely to indicate this fact. A mixture containing 100 mM Tris hydrochloride (pH 8.0), 5 mM MgCl 2 , 5 mM ATP, 5 mM DTT, 2 Ci [2-14 C]4-diphosphocytidyl-2C-methylerythritol (117 Ci͞mmol), 5 mM 4-diphosphocytidyl-2C-methylerythritol labeled with 13 C as indicated, and 0.1 mg of recombinant YchB protein from E. coli (16) in a total volume of 4 ml was incubated at 37°C. The reaction was monitored by 31 P NMR.…”

“…E. coli XL1-pQEygbB cells (2 g) were suspended in 30 ml of 100 mM Tris hydrochloride (pH 8.0) containing 20 mM imidazole hydrochloride and 500 mM NaCl (standard buffer). Cell extract was prepared as described earlier (16) and was applied to a column of Ni 2ϩ -chelating Sepharose FF (2 ϫ 8 cm; flow rate, 3 ml͞min) that had been equilibrated with standard buffer. The column was washed with 100 ml of standard buffer and developed with a linear gradient of 20-500 mM imidazole in standard buffer (total volume, 300 ml).…”

“…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.…”

Protein Interactome and Its Application to Protein Function Prediction

“…species), they are considered as functionally associated (Barker & Pagel, 2005;Bowers et al, 2004;Pellegrini et al, 1999). These methods applied to microorganisms and successfully discovered novel participants of known pathway (Carlson et al, 2004;Luttgen et al, 2000).…”