2-C-methyl-D-erythritol 4-phosphate Research Articles

Foliar treatment with MSB (menadione sodium bisulphite) to increase artemisinin content in Artemisia annua plants

Artemisinin is a sesquiterpene lactone endoperoxide, recognized for its application as a treatment for malaria. Besides, it has activity against several types of cancers, as well as anti-viral and anti-inflammatory properties. The chemical synthesis of artemisinin is quite complex and economically unfeasible. Hence, artemisinin is typically obtained from Artemisia annua plants in low quantities in relation to the biomass. A strategy to increase the biosynthesis of secondary metabolites would be the application of elicitors. In this work, the effect of treating A. annua plants with menadione sodium bisulphite (MSB) is studied. An initial dose-optimization was conducted by treating the plants with different MBS concentrations (0, 0.2, 0.8, 1, 2, 3 and 4 mM) and artemisinin was quantified 48 h after treatment. The highest artemisinin content (3.71 mg artemisinin g−1 DW) was obtained with 1 mM of MSB. Then, harvesting time was optimised (24, 48 and 72 h after treatment application) with 1 mM of MSB. The largest increase in artemisinin content was observed at 48 h with an increment of 62.37 % over the control. The treatment increased H2O2 content, as well as the activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Besides, MSB up-regulated HMGR (the gene for 3-hydroxy-3-methylglutaryl-CoA reductase) and DXS (the gene for 1-deoxy-D-xylulose-5-phosphate synthase) 24 h after treatment. Both genes are involved in the MVA (mevalonate) and MEP (2C-methylerythritol 4-phosphate) pathways, respectively. These pathways represent the initiation of artemisinin biosynthesis. Thus, MSB application and the consequent up-regulation of early biosynthetic pathway genes along with the triggered oxidative stress may have been contributed to the observed increase in artemisinin production.

An Insight Into Structure, Function, and Expression Analysis of 3-Hydroxy-3-Methylglutaryl-CoA Reductase of

Citronella ( Cymbopogon winterianus ) is one of the richest sources of high-value isoprenoid aromatic compounds used as flavour, fragrance, and therapeutic elements. These isoprenoid compounds are synthesized by 2 independent pathways: mevalonate pathway and 2-C-methyl-d-erythritol-4-phosphate pathway. Evidence suggests that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a rate-controlling enzyme for the synthesis of variety of isoprenoids. This study reports the isolation, characterization, and tissue-specific expression analysis of HMGR from citronella. The modelled HMGR is a class I type of HMGR enzyme with 3-domain architecture. The active site comprises a cofactor (nicotinamide adenine dinucleotide phosphate) and the substrate-binding motifs. The real-time and quantitative reverse transcription-polymerase chain reaction results revealed equal expression level in both leaf sheath and root tissue. The results from our study shall be a valuable resource for future molecular intervention to alter the metabolic flux towards improvement of key active ingredient in this important medicinal plant.

ChemInform Abstract: Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

AbstractReview: 145 refs.

Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids.

Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined.

Mimicking direct protein–protein and solvent-mediated interactions in the CDP-methylerythritol kinase homodimer: a pharmacophore-directed virtual screening approach

The 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate, which are the precursors of isoprenoids, is present in plants, in the malaria parasite Plasmodium falciparum and in most eubacteria, including pathogenic agents. However, the MEP pathway is absent from fungi and animals, which have exclusively the mevalonic acid pathway. Given the characteristics of the MEP pathway, its enzymes represent potential targets for the generation of selective antibacterial, antimalarial and herbicidal molecules. We have focussed on the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (CMK), which catalyses the fourth reaction step of the MEP pathway. A molecular dynamics simulation was carried out on the CMK dimer complex, and protein-protein interactions analysed, considering also water-mediated interactions between monomers. In order to find small molecules that bind to CMK and disrupt dimer formation, interactions observed in the dynamics trajectory were used to model a pharmacophore used in database searches. Using an intensity-fading matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry approach, one compound was found to interact with CMK. The data presented here indicate that a virtual screening approach can be used to identify candidate molecules that disrupt the CMK-CMK complex. This strategy can contribute to speeding up the discovery of new antimalarial, antibacterial, and herbicidal compounds.

Caterpillar Herbivory and Salivary Enzymes Decrease Transcript Levels of Medicago truncatula genes Encoding Early Enzymes in Terpenoid Biosynthesis

In response to caterpillar herbivory, alfalfa and related plant species defend themselves through the induction of saponin and volatile terpenoid biosynthesis. Both these types of defensive compounds are derived from the metabolic intermediate, isopentenyl diphosphate (IPP). In plants, two distinct biosynthetic pathways can generate IPP; the cytosolic mevalonate pathway and the plastid-associated 2C-methyl erythritol 4-phosphate (MEP) pathway. In Medicago truncatula, transcript levels of key regulatory genes active in the early steps of these biosynthetic pathways were measured in response to larval herbivory by the beet army worm, Spodoptera exigua. Transcripts encoding enzymes at early steps of both terpenoid pathways were lower in caterpillar-damaged leaves. Higher degrees of herbivore damage accentuated the decrease in transcript levels; however, transcript amounts were not affected by insect larval stage. Insect larvae, manipulated to reduce labial gland salivary secretions, were used to examine the role of the salivary elicitors in modulating gene expression. Results suggest that an insect salivary factor, possibly glucose oxidase (GOX), may be involved in reduction of transcript levels following herbivory. Addition of GOX or hydrogen peroxide to mechanically wounded leaves confirm these findings. In comparison, transcript levels of a gene encoding a putative terpene synthase are induced in mechanically- or insect-damaged leaves. These data show that insect salivary factors can act to suppress transcript levels of genes involved in plant defense pathways. Findings also suggest that in response to stress such as insect herbivory, regulation occurs at the early steps of the MEP pathway.

Biosynthetic Pathway of Insect Juvenile Hormone III in Cell Suspension Cultures of the Sedge Cyperus iria

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.

Biosynthetic Pathway of Insect Juvenile Hormone III in Cell Suspension Cultures of the Sedge Cyperus iria

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis inC. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P450 monooxygenase.

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

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.