Albert Boronat

Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids : a metabolic milestone achieved through genomics

Plants synthesize an enormous variety of metabolites that can be classified into two groups based on their function: primary metabolites, which participate in nutrition and essential metabolic processes within the plant, and secondary metabolites (also referred to as natural products), which

Expression and Molecular Analysis of the Arabidopsis DXR Gene Encoding 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase, the First Committed Enzyme of the 2- C -Methyl-d-Erythritol 4-Phosphate Pathway

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis. In Arabidopsis, DXR is encoded by a single-copy gene. We have cloned a full-length cDNA corresponding to this gene. A comparative analysis of all plant DXR sequences known to date predicted an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N terminus of the mature protein, which is not present in the prokaryotic DXR homologs. We demonstrate that Arabidopsis DXR is targeted to plastids and localizes into chloroplasts of leaf cells. The presence of the proline-rich region in the mature Arabidopsis DXR was confirmed by detection with a specific antibody. A proof of the enzymatic function of this protein was obtained by complementation of anEscherichia coli mutant defective in DXR activity. The expression pattern of β-glucuronidase, driven by theDXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR transcript and protein, revealed developmental and environmental regulation of theDXR gene. The expression pattern of theDXR gene parallels that of the Arabidopsis 1-deoxy-d-xylulose 5-phosphate synthase gene, but the former is slightly more restricted. These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and inflorescences. The block of the 2-C-methyl-d-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin led to a rapid accumulation of DXR protein, whereas the 1-deoxy-d-xylulose 5-phosphate synthase protein level was not altered. Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-d-erythritol 4-phosphate pathway.

Distinct Light-Mediated Pathways Regulate the Biosynthesis and Exchange of Isoprenoid Precursors during Arabidopsis Seedling Development

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.

The plastidial MEP pathway: unified nomenclature and resources

In plants, the plastid-localized 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway provides the precursors for the synthesis of isoprenoid hormones, monoterpenes, carotenoids and the side chain of chlorophylls, tocopherols and prenylquinones. As a result of the fast progress in the elucidation and characterization of the pathway (mainly by genetic approaches in Escherichia coli and Arabidopsis thaliana), different names have been used in the literature to designate the orthologous bacterial and plant genes and the corresponding null and partial loss-of-function mutants. This has led to a confusing variety of naming conventions in this field. Here, we propose a reorganization of the various naming systems with the aim of facilitating the dissemination and sharing of genetic resources and tools central to plant isoprenoid research.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen‐sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.

The Arabidopsis thaliana FPS1 Gene Generates a Novel mRNA That Encodes a Mitochondrial Farnesyl-diphosphate Synthase Isoform

The enzyme farnesyl-diphosphate synthase (FPS; EC2.5.1.1./EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. FPS is considered to play a key role in isoprenoid biosynthesis. We have reported previously that Arabidopsis thaliana contains two differentially expressed genes, FPS1 and FPS2, encoding two highly similar FPS isoforms, FPS1 and FPS2, (Cunillera, N., Arró, M., Delourme, D., Karst, F., Boronat, A., and Ferrer, A. (1996) J. Biol. Chem. 271, 7774–7780). In this paper we report the characterization of a novel ArabidopsisFPS mRNA (FPS1L mRNA) derived from the FPS1 gene. A cDNA corresponding to the FPS1L mRNA was cloned using a reverse transcription-polymerase chain reaction strategy. Northern blot analysis showed that the two FPS1-derived mRNAs are differentially expressed. The FPS1L mRNA accumulates preferentially in inflorescences, whereas the previously reported FPS1 mRNA (FPS1S mRNA) is predominantly expressed in roots and inflorescences. FPS1L mRNA contains an in-frame AUG start codon located 123 nucleotides upstream of the AUG codon used in the translation of the FPS1S isoform. Translation of the FPS1L mRNA from the upstream AUG codon generates a novel FPS1 isoform (FPS1L) with an NH2-terminal extension of 41 amino acid residues, which has all the characteristics of a mitochondrial transit peptide. The functionality of the FPS1L NH2-terminal extension as a mitochondrial transit peptide was demonstrated by its ability to direct a passenger protein to yeast mitochondria in vivo and by in vitro import experiments using purified plant mitochondria. TheArabidopsis FPS1L isoform is the first FPS reported to contain a mitochondrial transit peptide.

Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli.

An alternative mevalonate‐independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.

Isoprenoid Biosynthesis through the Methylerythritol Phosphate Pathway: The (E)‐4‐Hydroxy‐3‐methylbut‐2‐enyl Diphosphate Synthase (GcpE) is a [4Fe–4S] Protein

nitrilotriaceticacidagarosecolumn. The enzyme was found to be 95% pure by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electro-phoresis) and presented an apparent molecular mass of43kDa. The purified protein was inactive, even in thepresenceofthereducingsystemsdetailedbelow.Suchalackofcatalyticactivitywasprobablyaresultofthepredominant

Isolation and structural characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) catalyses the synthesis of mevalonate, the specific precursor of all isoprenoid compounds present in plants. We have characterized two overlapping cDNA clones that encompass the entire transcription unit of an HMG-CoA reductase gene from Arabidopsis thaliana. The transcription product has an upstream non-coding sequence of 70 nucleotides preceding an open reading frame of 1776 bases and a 3′ untranslated region in which two alternative polyadenylation sites have been found. The analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 592 residues with a molecular mass of 63 605 Da. The hydropathy profile of the protein indicates the presence of two highly hydrophobic domains near the N-terminus. A sequence of 407 amino acids corresponding to the C-terminal part of the protein (residues 172–579), which presumably contains the catalytic site, shows a high level of similarity to the region containing the catalytic site of the hamster, human, yeast and Drosophila enzymes. The N-terminal domain contains two putative membrane-spanning regions, in contrast to the enzyme from other organisms which has seven trans-membrane regions. A. thaliana contains two different HMG-CoA reductase genes (HMG1 and HMG2), as estimated by gene cloning and Southern blot analysis. Northern blot analysis reveals a single transcript of 2.4 kb in leaves and seedlings, which presumably corresponds to the expression of the HMG1 gene.