Santiago Imperial

Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis

Plastid isoprenoids are synthesized via the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway. A few years after its discovery, most of the Escherichia coli genes involved in the pathway have been identified, including gcpE. In this work, we have identified an Arabidopsis thaliana protein with homology to the product of this gene. The plant polypeptide, GCPE, contains two structural domains that are absent in the E. coli protein: an N‐terminal extension and a central domain of 30 kDa. We demonstrate that the N‐terminal region targets the Arabidopsis protein to chloroplasts in vivo, consistent with its role in plastid isoprenoid biosynthesis. Although the presence of the internal extra domain may have an effect on activity, the Arabidopsis mature GCPE was able to complement a gcpE‐defective E. coli strain, indicating the plant protein is a true functional homologue of the bacterial gcpE gene product.

Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase (GCPE) gene expression during carotenoid accumulation in ripening tomato fruit

Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato (Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe (r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.

An improved bradford protein assay for collagen proteins

A modification of the protein determination method of Bradford adapted for collagen-rich samples is described. The use of Coomassie-based protein determination methods is limited by the great variation in colour yield obtained for different proteins. This is especially important in samples containing significant amounts of collagen where direct application of the methods of Lowry and Bradford results in underestimated values. Addition of small amounts of sodium dodecyl sulphate (SDS) (0.0035%) to the diluted solutions of Coomassie Brilliant Blue G used as dye reagent in the Bradford colorimetric assay caused a 4-fold increase in the colour response of three collagen proteins (Col I, III and IV) and a decrease in absorbance for various non-collagen proteins. The presence of SDS in the reagent did not result in a significant metachromatic shift of the collagen-dye complexes. This simple modification in the preparation of the reagent for the Bradford assay allows similar response curves to be obtained for collagen and non-collagen proteins, making the modified assay of potential use for protein determination in collagen-rich samples such as pancreatic extracts.

The Methylerythritol Phosphate (MEP) Pathway for Isoprenoid Biosynthesis as a Target for the Development of New Drugs Against Tuberculosis

Tuberculosis remains a major infectious disease to humans. It accounts for approximately 8-9 million new cases worldwide and an estimated 1.6 million deaths annually. Effective treatments for tuberculosis consist of a combination of several drugs administered over long periods of time. Since Mycobacterium tuberculosis often acquires multiple drug resistant mechanisms, development of new drugs with innovative actions is urgently required. The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway, in charge of the essential biosynthesis of isoprenoids, represents a promising and selective target for developing new drugs against tuberculosis. To date, only fosmidomycin, a molecule that targets the second enzyme of the MEP pathway, has reached clinical trials but recent advances elucidating the structure and kinetics of the MEP enzymes are likely to change this scenario. This review describes the structure, mechanism of action and inhibitors of the seven enzymes of the MEP pathway, with special attention to the reported studies in M. tuberculosis.

Sources of interference in the use of 2,3-diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples

The use of 2,3-diaminonaphthalene (DAN) for the fluorimetric determination of nitric oxide synthase (NOS) activity in rat brain extracts has been re-examined. Two types of interference were observed, due either to components of the reaction mixture or to the enzymatic sample itself. One of the substrates (NADPH) and some cofactors (FADH(2), FMNH(2)) required for the enzyme activity interfere in the assay by quenching the fluorescence produced. Interference was minimized by using lower FADH(2), FMNH(2) and NADPH concentrations (1 micromol/l) and a NADPH recycling system in the reaction mixture. The addition of bovine serum albumin or hemoglobin to the sample quenched fluorescence intensity, but these protein interferences could be reduced by filtering the samples after reaction. We conclude that the DAN fluorimetric assay as originally described is not suitable for the determination of NOS activity in crude extracts such as rat brain cytosolic fraction, due to the presence of interfering substances. Nevertheless, DAN could be used for the determination of enzyme activity after reducing protein interference by filtering, or in less complex samples such as cell cultures (e.g. activated macrophages), or in chromatographic fractions obtained during the purification of the enzyme. A careful use of the commercial kits based on the use of DAN for the determination of NOS activity is recommended.

A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2‐C‐methyl‐d‐erythritol‐4‐phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single‐molecule force spectroscopy (SMFS), we have probed at the single‐molecule level the interaction of 1‐deoxy‐d‐xylulose 5‐phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde‐3‐phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single‐molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 µM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2‐step process with dissociation constants of koff = 6.1 × 10−4 ± 7.5 × 10−3 and 1.3 × 10−2 ± 1.0 × 10−2 s−1, and reaction lengths of xβ = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.—Sisquella, X., de Pourcq, K., Alguacil, J., Robles, J., Sanz, F., Anselmetti, D., Imperial, S., Fernàndez‐Busquets, X. A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials. FASEB J. 24, 4203–4217 (2010). www.fasebj.org

Biosynthesis of isoprenoids in plants: Structure of the 2C-methyl-D-erithrytol 2,4-cyclodiphosphate synthase from Arabidopsis thaliana. Comparison with the bacterial enzymes

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 Å 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.

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.

Enzymatic synthesis of 1-deoxysugar-phosphates using E. coli 1-deoxy-d-xylulose 5-phosphate synthase

Abstract The thiamine diphosphate-dependent enzyme 1-deoxy- d -xylulose 5-phosphate synthase from E. coli can use d -erythrose 4-phosphate and d -ribose 5-phosphate as alternative substrates. These reactions were used for the production of 1-deoxy- d -fructose 6-phosphate and 1-deoxy- d -sedoheptulose 7-phosphate and have potential application for the biosynthesis of other 1-deoxysugar phosphates.

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.