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

ABSTRACT: A hypothetical gene with similarity to the ispD gene of Escherichia coli was cloned from Arabidopsis thaliana cDNA. The ORF of 909 bp specifies a protein of 302 amino acid residues. The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78-202 bp. A fragment comprising amino acid residues 76-302 was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl-d-erythritol from 2C-methyl-d-erythritol 4-phosphate with a specific activity of 67 micromol small middle dotmin(-1) mg(-1). The Michaelis constants for 4-diphosphocytidyl-2C-methyl-d-erythritol and CTP were 500 microM and 114 microM, respectively.

SUBMITTER: Rohdich F

PROVIDER: S-EPMC18623 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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

Rohdich F F Wungsintaweekul J J Eisenreich W W Richter G G Schuhr C A CA Hecht S S Zenk M H MH Bacher A A

Proceedings of the National Academy of Sciences of the United States of America 20000601 12

A hypothetical gene with similarity to the ispD gene of Escherichia coli was cloned from Arabidopsis thaliana cDNA. The ORF of 909 bp specifies a protein of 302 amino acid residues. The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78-202 bp. A fragment comprising amino acid residues 76-302 was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl-d-e ...[more]

PMID: 10841550

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

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Project description:The X-ray crystal structure of the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 A resolution in complex with a cytidine-5-monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate-like binding pocket, with a proposed feedback-regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.

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Structure of 2C-methyl-D-erythritol 2,4- cyclodiphosphate synthase: an essential enzyme for isoprenoid biosynthesis and target for antimicrobial drug development.

Project description:The crystal structure of the zinc enzyme Escherichia coli 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase in complex with cytidine 5'-diphosphate and Mn(2+) has been determined to 1.8-A resolution. This enzyme is essential in E. coli and participates in the nonmevalonate pathway of isoprenoid biosynthesis, a critical pathway present in some bacterial and apicomplexans but distinct from that used by mammals. Our analysis reveals a homotrimer, built around a beta prism, carrying three active sites, each of which is formed in a cleft between pairs of subunits. Residues from two subunits recognize and bind the nucleotide in an active site that contains a Zn(2+) with tetrahedral coordination. A Mn(2+), with octahedral geometry, is positioned between the alpha and beta phosphates acting in concert with the Zn(2+) to align and polarize the substrate for catalysis. A high degree of sequence conservation for the enzymes from E. coli, Plasmodium falciparum, and Mycobacterium tuberculosis suggests similarities in secondary structure, subunit fold, quaternary structure, and active sites. Our model will therefore serve as a template to facilitate the structure-based design of potential antimicrobial agents targeting two of the most serious human diseases, tuberculosis and malaria.

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Project description:2-C-methyl-D-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the precursor for the synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME), another key intermediate of the non-mevalonate pathway. As this pathway is non-existent in higher animals, including humans, it represents great opportunities for novel antimicrobial development. To facilitate the in-depth studies of this pathway, we reported here a formal synthesis of CDP-ME through a new synthesis of 2-C-Methyl-D-erythritol-4-phosphoric acid from D-(+)-arabitol.

| S-EPMC3462025 | biostudies-literature

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