Biosynthesis of Terpenoids: YchB Protein of Escherichia coli Phosphorylates the 2-hydroxy Group of 4-diphosphocytidyl-2C-methyl-D-erythritol

• Published in: Proceedings of the National Academy of Sciences - PNAS 2000-02, Vol.97 (3), p.1062-1067
• Main Authors: Luttgen, Holger, Rohdich, Felix, Herz, Stefan, Wungsintaweekul, Juraithip, Hecht, Stefan, Schuhr, Christoph A., Fellermeier, Monika, Sagner, Sylvia, Zenk, Meinhart H., Bacher, Adelbert, Eisenreich, Wolfgang
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteria; Bacteria - genetics; Bacterial Proteins - metabolism; Biochemistry; Biological Sciences; Biosynthesis; Capsicum - metabolism; Chemical equilibrium; Chromoplasts; Enzymes; Erythritol - analogs & derivatives; Erythritol - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Genes; Genomes; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phosphates; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Plants - genetics; Plants, Medicinal; Plastids - metabolism; Polyisoprenyl Phosphates - biosynthesis; Proteins; Radioactive decay; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Spectroscopy; Terpenoids; Xylulose - analogs & derivatives; Xylulose - metabolism; YchB protein
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.3.1062

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 various13C-labeled substrate samples. A14C-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.