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Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from tomato.

ABSTRACT: The putative catalytic domain (residues 81-401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 micromol small middle dotmg(-1) small middle dotmin(-1). The structure of the reaction product, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate, was established by NMR spectroscopy. Divalent metal ions, preferably Mg(2+), are required for activity. Neither the tomato enzyme nor the E. coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.

SUBMITTER: Rohdich F

PROVIDER: S-EPMC26933 | biostudies-literature | 2000 Jul

REPOSITORIES: biostudies-literature

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Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from tomato.

Rohdich F F Wungsintaweekul J J Luttgen H H Fischer M M Eisenreich W W Schuhr C A CA Fellermeier M M Schramek N N Zenk M H MH Bacher A A

Proceedings of the National Academy of Sciences of the United States of America 20000701 15

The putative catalytic domain (residues 81-401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 micromol small middle dotmg(-1) small middle dotmin(-1). The structure of the reaction product, 4-diphosph ...[more]

PMID: 10880567

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

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A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C-methyl-D-erythritol kinase and reassessment of the quaternary structure.

Project description:4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE; EC 2.7.1.148) contributes to the 1-deoxy-D-xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C-methyl-D-erythritol. A triclinic crystal structure of the Escherichia coli IspE-ADP complex with two molecules in the asymmetric unit was determined at 2 A resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the ;triclinic dimer' is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization.

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Identification of novel small molecule inhibitors of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase of Gram-negative bacteria.

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