A cytosolic bifunctional geranyl/farnesyl diphosphate synthase provides MVA-derived GPP for geraniol biosynthesis in rose flowers.

Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis "Old Blush" flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-ß-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.

Divergent contribution of the MVA and MEP pathways to the formation of polyprenols and dolichols in Arabidopsis.

Isoprenoids, including dolichols (Dols) and polyprenols (Prens), are ubiquitous components of eukaryotic cells. In plant cells, there are two pathways that produce precursors utilized for isoprenoid biosynthesis: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. In this work, the contribution of these two pathways to the biosynthesis of Prens and Dols was addressed using an in planta experimental model. Treatment of plants with pathway-specific inhibitors and analysis of the effects of various light conditions indicated distinct biosynthetic origin of Prens and Dols. Feeding with deuteriated, pathway-specific precursors revealed that Dols, present in leaves and roots, were derived from both MEP and MVA pathways and their relative contributions were modulated in response to precursor availability. In contrast, Prens, present in leaves, were almost exclusively synthesized via the MEP pathway. Furthermore, results obtained using a newly introduced here 'competitive' labeling method, designed so as to neutralize the imbalance of metabolic flow resulting from feeding with a single pathway-specific precursor, suggest that under these experimental conditions one fraction of Prens and Dols is synthesized solely from endogenous precursors (deoxyxylulose or mevalonate), while the other fraction is synthesized concomitantly from endogenous and exogenous precursors. Additionally, this report describes a novel methodology for quantitative separation of 2H and 13C distributions observed for isotopologues of metabolically labeled isoprenoids. Collectively, these in planta results show that Dol biosynthesis, which uses both pathways, is significantly modulated depending on pathway productivity, while Prens are consistently derived from the MEP pathway.

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives.

Isoprenoids, a diverse class of natural products, are present in all living organisms. Their two universal building blocks are synthesized via two independent pathways: the mevalonate pathway and the 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) pathway. The presence of the latter in pathogenic bacteria and its absence in humans make all its enzymes suitable targets for the development of novel antibacterial drugs. (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), the last intermediate of this pathway, is a natural ligand for the human Vγ9Vδ2 T cells and the most potent natural phosphoantigen known to date. Moreover, 5-hydroxypentane-2,3-dione, a metabolite produced by Escherichia coli 1-deoxy-ᴅ-xylulose 5-phosphate synthase (DXS), the first enzyme of the MEP pathway, structurally resembles (S)-4,5-dihydroxy-2,3-pentanedione, a signal molecule implied in bacterial cell communication. In this review, we shed light on the diversity of potential uses of the MEP pathway in antibacterial therapies, starting with an overview of the antibacterials developed for each of its enzymes. Then, we provide insight into HMBPP, its synthetic analogs, and their prodrugs. Finally, we discuss the potential contribution of the MEP pathway to quorum sensing mechanisms. The MEP pathway, providing simultaneously antibacterial drug targets and potent immunostimulants, coupled with its potential role in bacterial cell-cell communication, opens new therapeutic perspectives.

New Application of cycloSaligenyl Prodrugs Approach for the Delivery of Fosfoxacin Derivatives in Mycobacteria.

In this work, we implemented for the first time the cycloSaligenyl prodrug strategy to increase the bioavailability of fosmidomycin phosphate analogs in bacteria. Here, we report the synthesis of 34 cycloSaligenyl prodrugs of fosfoxacin and its derivatives. Among them, fifteen double prodrugs efficiently prevented the growth of the non-pathogenic, fast-growing Mycobacterium smegmatis.

Biochemical and Mutational Analysis of Radical S-Adenosyl-L-Methionine Adenosylhopane Synthase HpnH from Zymomonas mobilis Reveals that the Conserved Residue Cysteine-106 Reduces a Radical Intermediate and Determines the Stereochemistry.

Adenosylhopane is a crucial precursor of C35 hopanoids, which are believed to modulate the fluidity and permeability of bacterial cell membranes. Adenosylhopane is formed by a crosslinking reaction between diploptene and a 5'-deoxyadenosyl radical that is generated by the radical S-adenosyl-L-methionine (SAM) enzyme HpnH. We previously showed that HpnH from Streptomyces coelicolor A3(2) (ScHpnH) converts diploptene to (22R)-adenosylhopane. However, the mechanism of the stereoselective C-C bond formation was unclear. Thus, here, we performed biochemical and mutational analysis of another HpnH, from the ethanol-producing bacterium Zymomonas mobilis (ZmHpnH). Similar to ScHpnH, wild-type ZmHpnH afforded (22R)-adenosylhopane. Conserved cysteine and tyrosine residues were suggested as possible hydrogen sources to quench the putative radical reaction intermediate. A Cys106Ala mutant of ZmHpnH had one-fortieth the activity of the wild-type enzyme and yielded both (22R)- and (22S)-adenosylhopane along with some related byproducts. Radical trapping experiments with a spin-trapping agent supported the generation of a radical intermediate in the ZmHpnH-catalyzed reaction. We propose that the thiol of Cys106 stereoselectively reduces the radical intermediate generated at the C22 position by the addition of the 5'-deoxadenosyl radical to diploptene, to complete the reaction.

α,α-Difluorophosphonohydroxamic Acid Derivatives among the Best Antibacterial Fosmidomycin Analogues.

Three α,α-difluorophosphonate derivatives of fosmidomycin were synthesized from diethyl 1,1-difluorobut-3-enylphosphonate and were evaluated on Escherichia coli. Two of them are among the best 1-deoxy-d-xylulose 5-phosphate reductoisomerase inhibitors, with IC50 in the nM range, much better than fosmidomycin, the reference compound. They also showed an enhanced antimicrobial activity against E. coli on Petri dishes in comparison with the corresponding phosphates and the non-fluorinated phosphonate.

Characterization of Radical SAM Adenosylhopane Synthase, HpnH, which Catalyzes the 5'-Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C35 Bacteriohopanepolyols.

Adenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S-adenosyl-l-methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)-adenosylhopane, indicating that HpnH catalyzes stereoselective C-C formation between C29 of diploptene and C5' of 5'-deoxyadenosine. Further, the HpnH reaction in D2 O-containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C35 bacteriohopane skeleton.

Synthesis and biological evaluation of aryl phosphoramidate prodrugs of fosfoxacin and its derivatives.

Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized and investigated for their ability to target bacteria. No growth inhibition was observed neither for Mycobacterium smegmatis nor for Escherichia coli on solid medium, demonstrating the absence of release of the active compounds in the bacterial cells. Investigation of the stability of the prodrugs and their multienzymatic cleavage in abiotic and biotic conditions showed that the use of aryl phosphoramidate prodrug approach to deliver non-nucleotides compounds is not obvious and might not be appropriate for an antimicrobial drug.

Biosynthesis of Isoprene Units in Euphorbia lathyris Laticifers vs. Other Tissues: MVA and MEP Pathways, Compartmentation and Putative Endophytic Fungi Contribution.

Euphorbia species are characterized by a net of laticifers producing large amounts of triterpenes. These hydrocarbon-like metabolites can be converted into fuel by the methods of the oil industry. Euphorbia lathyris is easily grown at an industrial scale. In an attempt to increase its triterpene production, the metabolic pathways leading to isoprenoid were investigated by incorporation of 13C labeled glucose and mevalonate and 2H labeled deoxyxylulose as well as by natural abundance isotope ratio GC-MS. Latex triterpenes are exclusively synthesized via the mevalonate (MVA) pathway: this may orient future search for improving the triterpene production in E. lathyris. Phytosterols and their precursors are mainly derived from MVA pathway with a slight contribution of the methylerythritol phosphate (MEP) pathway, whereas phytol is issued from MEP pathway with a minor contribution of the MVA pathway: this is in accordance with the metabolic cross-talk between cytosolic and plastidial compartments in plants. In addition, hopenol B behaved differently from the other latex triterpenes. Its 13C isotope abundance after incorporation of 13C labeled glucose and its natural abundance δ2H signature clearly differed from those of the other latex triterpenes indicating another metabolic origin and suggesting that it may be synthesized by an endophytic fungus.

Synthesis and biological evaluation of phosphate isosters of fosmidomycin and analogs as inhibitors of Escherichia coli and Mycobacterium smegmatis 1-deoxyxylulose 5-phosphate reductoisomerases.

Hydroxamate analogs of fosfoxacin, the phosphate homolog of fosmidomycin, have been synthesized and their activity tested on Escherichia coli and Mycobacterium smegmatis DXRs. Except for compound 4b, the IC50 values of phosphate derivatives are approximately 10-fold higher than those of the corresponding phosphonates. Although their inhibitory activity on Escherichia coli DXR is less efficient than their phosphonate analogs, we report the ability of phosphate compounds to inhibit the growth of Escherichia coli. This work points out that the uptake of fosfoxacin and its analogs is taking place via the GlpT and UhpT transporters. As expected, these compounds are inefficient to inhibit the growth of M. smegmatis growth inhibition probably due to a lack of uptake.

C35 Hopanoid Side Chain Biosynthesis: Reduction of Ribosylhopane into Bacteriohopanetetrol by a Cell-Free System Derived from Methylobacterium organophilum.

The major bacterial triterpenoids of the hopane series each consist of a C30 triterpene hopane moiety and an additional nonterpene C5 side chain derived from D-ribose and linked through its C-5 carbon atom to the hopane side chain. Bacteriohopanetetrol and aminobacteriohopanetriol are the most common representatives of this natural product series, adenosylhopane and ribosylhopane being putative precursors. Deuterium-labelled ribosylhopane was obtained by hemisynthesis and converted into deuterium-labelled bacteriohopanetetrol in the presence of NADPH, thus giving evidence of this as yet unknown precursor-to-product relationship in the bacterial hopanoid metabolic pathway.

S-carvone suppresses cellulase-induced capsidiol production in Nicotiana tabacum by interfering with protein isoprenylation.

S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.

Hemisynthesis of deuteriated adenosylhopane and conversion into bacteriohopanetetrol by a cell-free system from Methylobacterium organophilum.

Adenosylhopane is a putative precursor of the widespread bacterial C35 biohopanoids. A concise and flexible hemisynthesis of adenosylhopane has been developed including as key steps a cross metathesis between two olefins containing either the hopane moiety or a protected adenosine derivative and a subsequent diimide reduction of the resulting olefin. Reduction by deuteriated diimide allowed deuterium labelling. This synthetic protocol represents a versatile tool to access to deuteriated composite bacterial hopanoids required for biosynthetic studies. Deuteriated adenosylhopane was thus converted into bacteriohopanetetrol by a crude cell-free system from Methylobacterium organophilum in the presence of NADPH, showing for the first time the precursor to product relationship between these two bacterial metabolites.

Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase.

Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC50 values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.

Ribosylhopane, a novel bacterial hopanoid, as precursor of C35 bacteriohopanepolyols in Streptomyces coelicolor A3(2).

Wild-type Streptomyces coelicolor A3(2) produces aminobacteriohopanetriol as the only elongated C35 hopanoid. The hopanoid phenotype of two mutants bearing a deletion of genes from a previously identified hopanoid biosynthesis gene cluster provides clues to the formation of C35 bacteriohopanepolyols. orf14 encodes a putative nucleosidase; its deletion induces the accumulation of adenosylhopane as it cannot be converted into ribosylhopane. orf18 encodes a putative transaminase; its deletion results in the accumulation of adenosylhopane, ribosylhopane, and bacteriohopanetetrol. Ribosylhopane was postulated twenty years ago as a precursor for bacterial hopanoids but was never identified in a bacterium. Absence of the transaminase encoded by orf18 prevents the reductive amination of ribosylhopane into aminobacteriohopanetriol and induces its accumulation. Its reduction by an aldose-reductase-like enzyme produces bacteriohopanetetrol, which is normally not present in S. coelicolor.

Catechol-rhodanine derivatives: Specific and promiscuous inhibitors of Escherichia coli deoxyxylulose phosphate reductoisomerase (DXR).

To develop more effective inhibitors than fosmidomycin, a natural compound which inhibits the deoxyxylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway, we designed molecules possessing on the one hand a catechol that is able to chelate the magnesium dication and on the other hand a group able to occupy the NADPH recognition site. Catechol-rhodanine derivatives (1-6) were synthesized and their potential inhibition was tested on the DXR of Escherichia coli. For the inhibitors 1 and 2, the presence of detergent in the enzymatic assays led to a dramatic decrease of the inhibition suggesting, that these compounds are rather promiscuous inhibitors. The compounds 4 and 5 kept their inhibition capacity in the presence of Triton X100 and could be considered as specific inhibitors of DXR. Compound 4 showed antimicrobial activity against Escherichia coli. The only partial protection of NADPH against the inhibition suggested that the catechol-rhodanine derivatives did not settle in the coenzyme binding site. This paper points out the necessity to include a detergent in the DXR enzymatic assays to avoid false positive when putative hydrophobic inhibitors are tested and especially when the IC50, are in the micromolar range.

Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the methylerythritol phosphate pathway.

The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K(i) = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K(i) = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.

Synthesis of tetrazole analogues of phosphonohydroxamic acids: an attempt to improve the inhibitory activity against the DXR.

This work is focused on the design of new antimicrobial drugs and on the development of lipophilic inhibitors of the DXR, the second enzyme of the MEP pathway for the biosynthesis of isoprene units in most bacteria, by replacing the phosphonate group of fosmidomycin derivatives by a tetrazoyl moiety capable of multiple hydrogen bonding. The N- and C-substituted tetrazole analogues of phosphonohydroxamate inhibitors were synthesized and tested on the DXR of Escherichia coli. This work points out the hypothesis that the phosphonate/phosphate recognition site might be too rigid to accommodate other functional groups.

Chemical constituents from Glyphaea brevis and Monodora myristica: chemotaxonomic significance.

A chemical investigation of the Glyphaea brevis leaves and of the Monodora myristica fruits led to the identification of thirteen compounds, seven linear long-chain aliphatic compounds, 1, 2, 4, 6, and 9-11, three steroids, 3a, 3b, and 7, two triterpenes, 5a and 5b, and one polyol, 8. The compounds 2 and 8, previously mentioned in the literature, are here characterized by their complete (1)H- and (13)C-NMR assignments. This is the first report of a full NMR assignment for linear fatty acid esters of aliphatic primary alcohols and for meso-erythritol. Compound 5b and 8 were isolated for the first time from plant extracts of the Tiliaceae family, and compounds 9-11 from the Annonaceae plant family. Our results constitute the basis for further chemotaxonomic studies on the two species.

Modifications around the hydroxamic acid chelating group of fosmidomycin, an inhibitor of the metalloenzyme 1-deoxyxylulose 5-phosphate reductoisomerase (DXR).

Fosmidomycin derivatives in which the hydroxamic acid group has been replaced by several bidentate chelators as potential hydroxamic alternatives were prepared and tested against the DXR from Escherichia coli. These results illustrate the predominant role of the hydroxamate functional group as the most effective metal binding group in DXR inhibitors.