Non-mevalonate Pathway Of Isoprenoid Biosynthesis Research Articles

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

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.

Crystal Structure of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase, a Crucial Enzyme in the Non-mevalonate Pathway of Isoprenoid Biosynthesis

We have solved the 2.5-A crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp(150), Glu(152), Glu(231), and Glu(234) are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed.

The lytB gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesis.

LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

Biosynthesis of terpenoids. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) from Plasmodium falciparum.

The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase was expressed in a recombinant E. coli strain. The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate at a rate of 4.3 micromol x mg(-1) x min(-1). At lower rates, the recombinant protein catalyzes the formation of 2-phospho-2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate and the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol. Divalent metal ions such as magnesium or manganese are required for catalytic activity. The enzyme has a pH optimum at pH 7.0. Recombinant expression of the full-length open reading frame afforded insoluble protein that could not be folded in vitro. The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis.

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.

Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis.

It is proposed that the lytB gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of lytB were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The Synechocystis lytB gene and a lytB cDNA from the flowering plant Adonis aestivalis were each found to significantly enhance accumulation of carotenoids in Escherichia coli engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (dxs), the initial enzyme of the DOXP pathway, the individual salutary effects of lytB and dxs were multiplied. In contrast, the combination of lytB and a cDNA encoding IPP isomerase (ipi) was no more effective in enhancing carotenoid accumulation than ipi alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.

Biosynthesis of Isoprenoids. A Rapid Method for the Preparation of Isotope-Labeled 4-Diphosphocytidyl-2C-methyl-d-erythritol

4-Diphosphocytidyl-2C-methyl-d-erythritol serves as an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis. The compound has been prepared in millimole quantity by a sequence of one-pot reactions using 13C-labeled pyruvate and dihydroxyacetone phosphate or 13C-labeled glucose as starting materials and recombinant enzymes of the nonmevalonate isoprenoid pathway as catalysts. The method has been used for the preparation of various 4-diphosphocytidyl-2C-methyl-d-erythritol isotopomers in high yield.

Temperature-sensitive Arabidopsis mutant defective in 1-deoxy- d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis

Temperature-sensitive Arabidopsis mutant defective in 1-deoxy- d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis

Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

Biosynthesis of [formula omitted], a putative C 5 intermediate in the mevalonate independent pathway for isoprenoid biosynthesis

According to incorporations of 13 labelled glucose, 2-C- methyl- d-erythritol was most likely synthesized by Corynebacterium ammoniagenes from glyceraldehyde 3-phosphate and from pyruvate via a condensation and a rearrangement. The observed labelling patterns for methylerythritol were identical with those of the isoprenic units of dihydromenaquinones from the same bacterium, suggesting for this tetrol an intermediate role in the non-mevalonate pathway for isoprenoid biosynthesis.

Glyceraldehyde 3-Phosphate and Pyruvate as Precursors of Isoprenic Units in an Alternative Non-mevalonate Pathway for Terpenoid Biosynthesis

Incorporation of 13C-labeled glycerol or pyruvate into the ubiquinone Q8 of Escherichia coli mutants lacking enzymes of the triose phosphate metabolism and of (U-13C6)glucose into the triterpenoids of the hopane series of Zymomonas mobilis showed that glyceraldehyde 3-phosphate (or eventually glyceraldehyde) and a C2 unit derived from pyruvate decarboxylation were the only precursors of the C5 skeleton of isoprenic units in a novel non-mevalonate pathway for isoprenoid biosynthesis in these bacteria.