(-)-2C-methyl-D-erythrono-1,4-lactone is formed after application of the terpenoid precursor 1-deoxy-D-xylulose.

Application of [1,2-14C]1-deoxy-D-xylulose, the committed precursor of terpenoids, thiamine and pyridoxol, to a variety of plant species resulted in the labelling of an unknown metabolite. The isolation and purification of this metabolite from Ipomoea purpurea plants fed with 1-deoxy-D-xylulose (DX), followed by NMR analysis, resulted in the identification of its structure as (-)-2C-methyl-D-erythrono-1,4-lactone (MDEL). MDEL has been previously isolated as a stress metabolite of certain plants. A hypothetical biosynthetic scheme is given.

Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata.

The nickel content in different parts of the hyperaccumulating tree Sebertia acuminata was analysed by atomic absorption spectroscopy. Nickel was found to be mainly located in laticifers. The total nickel content of a single mature tree was estimated to be 37 kg. By gel filtration and NMR spectroscopy, citric acid was unequivocally identified as counter ion for about 40% of this metal present. Nitrate was assumed to be a further partner for a complete ionic balance. Phytochelatins were not found to be involved in nickel detoxification in Sebertia. The localization of nickel complexes inside the laticifers was demonstrated by light microscopy as well as by scanning electron microscopy in combination with an EDX system for the analysis of elements. A repellent effect of the plant sap was observed on the fruit fly Drosophila melanogaster indicating that in hyperaccumulating plants nickel functions as an agent to prevent predation.

Endothelin in septic patients: effects on cardiovascular and renal function and its relationship to proinflammatory cytokines.

OBJECTIVE: To determine the time course of big-endothelin (big-ET) and its relationship to proinflammatory cytokines and organ function in sepsis. DESIGN: Prospective analysis in patients meeting criteria of severe sepsis as part of a multicenter study (RAMSES) with an anti-tumor necrosis factor monoclonal antibody F(ab')2 fragment (afelimomab). SETTING: University hospital intensive care unit. PATIENTS: A total of 23 nontrauma patients with severe sepsis or septic shock and ten multiple trauma patients. Septic patients were randomized for additional experimental treatment when initial interleukin (IL)-6 serum level was above 1000 pg/mL. INTERVENTIONS: Randomized patients received 1.0 mg/kg afelimomab or placebo three times daily over 3 days in addition to standard treatment. In each patient, serial blood samples for plasma big-ET and cytokine determination as well as clinical data were collected over 28 days. MEASUREMENTS AND MAIN RESULTS: Significantly increased concentrations of circulating big-ET were found in patients with severe sepsis as compared with healthy subjects. In septic patients, big-ET plasma levels were higher than in multiple trauma patients, and were more elevated in randomized than in nonrandomized patients. At study entry (day 0), big-ET reached a peak concentration and significantly correlated with IL-6 (r2 = .43) and IL-8 (r2 = .44) in patients with severe sepsis. Moreover, big-ET levels in septic patients, pooled over all observation days, correlated positively with pressure-adjusted heart rate, central venous pressure, pulmonary artery pressure, and pulmonary vascular resistance and correlated inversely with creatinine clearance (r2 = .54, .54, .59, .40, and .51, respectively, p = .0001). In all randomized septic patients, pressure-adjusted heart rate decreased from day 0 to day 2 in parallel with big-ET; however, a significant decrease in big-ET (day 0 to day 2) was only found in patients additionally treated with afelimomab. CONCLUSIONS: In patients with severe sepsis, big-ET plasma levels are markedly increased, even above those of multiple trauma patients, in close relationship to IL-6 and IL-8, and with significant correlation to renal function and pulmonary vascular tone.

Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular skeletal rearrangement.

The incorporation of [1-13C]- and [2,3,4,5-13C4]1-deoxy-D-xylulose into beta-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13C Enrichment and 13C13C coupling patterns showed conclusively that 1-deoxy-D-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, beta-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

Dimethylallyl pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants.

Cell cultures of Catharanthus roseus were supplied with [2-13C, 3-2H]-deoxyxylulose or [2-13C,4-2H]1-deoxyxylulose. Lutein and chlorophylls were isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded phytol. Isotope labeling patterns of phytol and lutein were determined by 2H NMR and 1H,2H-decoupled 13C NMR. From the data it must be concluded that the deuterium atom in position 3 of deoxyxylulose was incorporated into both isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate with a rate of 75% (with respect to the internal 13C label). The detected stereochemical signature implies that the label is located preferentially in the (E)-hydrogen atom of IPP. This preferential labeling, in turn, rules out dimethylallyl pyrophosphate as the compulsory precursor of IPP. In the experiment with [2-13C, 4-2H]1-deoxyxylulose, the 13C label was efficiently transferred to the terpenoids whereas the 2H label was completely washed out, most probably after IPP formation as a consequence of the isomerization and elongation process. In addition, the data cast light on the stereochemical course of the dehydrogenation and cyclization steps involved in the biosynthesis of lutein.

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

In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP, which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using (13)C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn(2+) or Mg(2+) but no other cofactors. Radioactivity from [2-(14)C]2C-methyl-D-erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol into 2C-methyl-D-erythritol 3,4-cyclophosphate. This compound does not serve as substrate for the formation of carotenoids by isolated chromoplasts and is assumed to be an in vitro product without metabolic relevance.

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

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.

Cytidine 5'-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol.

2-C-methylerythritol 4-phosphate has been established recently as an intermediate of the deoxyxylulose phosphate pathway used for biosynthesis of terpenoids in plants and in many microorganisms. We show that an enzyme isolated from cell extract of Escherichia coli converts 2-C-methylerythritol 4-phosphate into 4-diphosphocytidyl-2-C-methylerythritol by reaction with CTP. The enzyme is specified by the hitherto unannotated ORF ygbP of E. coli. The cognate protein was obtained in pure form from a recombinant hyperexpression strain of E. coli harboring a plasmid with the ygbP gene under the control of a T5 promoter and lac operator. By using the recombinant enzyme, 4-diphosphocytidyl-[2-(14)C]2-C-methylerythritol was prepared from [2-(14)C]2-C-methylerythritol 4-phosphate. The radiolabeled 4-diphosphocytidyl-2-C-methylerythritol was shown to be efficiently incorporated into carotenoids by isolated chromoplasts of Capsicum annuum. The E. coli ygbP gene appears to be part of a small operon also comprising the unannotated ygbB gene. Genes with similarity to ygbP and ygbB are present in the genomes of many microorganisms, and their occurrence appears to be correlated with that of the deoxyxylulose pathway of terpenoid biosynthesis. Moreover, several microorganisms have genes specifying putative fusion proteins with ygbP and ygbB domains, suggesting that both the YgbP protein and the YgbB protein are involved in the deoxyxylulose pathway. A gene from Arabidopsis thaliana with similarity to ygbP carries a putative plastid import sequence, which is well in line with the assumed localization of the deoxyxylulose pathway in the plastid compartment of plants.

Studies on the nonmevalonate pathway of terpene biosynthesis. The role of 2C-methyl-D-erythritol 2,4-cyclodiphosphate in plants.

2C-methyl-D-erythritol 2,4-cyclodiphosphate was recently shown to be formed from 2C-methyl-D-erythritol 4-phosphate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynthesis. To complement previous work with radiolabelled precursors, we have now demonstrated that [U-13C5]2C-methyl-D-erythritol 4-phosphate affords [U-13C5]2C-methyl-D-erythritol 2,4-cyclodiphosphate in isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplasts are shown to efficiently convert 2C-methyl-D-erythritol 4-phosphate as well as 2C-methyl-D-erythritol 2,4-cyclodiphosphate into the carotene precursor phytoene. The bulk of the kinetic data collected in competition experiments with radiolabeled substrates is consistent with the notion that the cyclodiphosphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2'-13C2]2C-methyl-D-erythritol 2,4-cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase.