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.

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.

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.

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.

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.

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation.

We have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

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.

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.

The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells.

We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

Plant isoprenoid biosynthesis via the MEP pathway: in vivo IPP/DMAPP ratio produced by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase in tobacco BY-2 cell cultures.

Feeding tobacco BY-2 cells with [2-(13)C,4-(2)H]deoxyxylulose revealed from the (13)C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% (2)H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. (2)H retention characterizes the isoprene units derived from the DMAPP branch, whereas (2)H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase, the IPP isomerase and the trans-prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with (2)H labeled deoxyxylulose.

Isoprenoid biosynthesis via the MEP pathway: in vivo Mössbauer spectroscopy identifies a [4Fe-4S]2+ center with unusual coordination sphere in the LytB protein.

The MEP pathway for the biosynthesis of isoprene units is present in most pathogenic bacteria, in the parasite responsible for malaria, and in plant plastids. This pathway is absent in animals and is accordingly a target for the development of antimicrobial drugs. LytB, also called IspH, the last enzyme of this pathway catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using an oxygen sensitive iron sulfur cluster. The exact nature of this iron sulfur cluster is still a matter of debate. We have used (57)Fe Mössbauer spectroscopy to investigate the LytB cluster in whole E. coli cells and in the anaerobically purified enzyme: In LytB an unusual [4Fe-4S](2+) cluster is attached to the protein by three conserved cysteines and contains a hexacoordinated iron linked to three sulfurs of the cluster and three additional oxygen or nitrogen ligands.

The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

Rapid structural elucidation of composite bacterial hopanoids by atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry.

Bacteriohopanepolyols (BHPs) are membrane lipids produced by a wide range of eubacteria. Their use, however, as molecular markers of bacterial populations and processes has until recently been hampered by the lack of a suitable rapid method for fingerprinting their composition in complex environmental matrices. New analytical procedures employing ion trap mass spectrometry now allow us to investigate the occurrence of BHPs in diverse biological and environmental samples including bacterial cultures, soils, and recent and ancient sediments. Here, we describe the structural characterisation using atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry (APCI-LC/MS(n)) of a number of previously identified but less commonly occurring BHPs such as adenosylhopane and ribonylhopane. Many of the structures described here have previously only been reported in one or just a small number of cultured organisms having been isolated from large amounts of cellular mass (4-26 g) and identified by nuclear magnetic resonance (NMR) techniques after purification of individual compounds. Now, having established characteristic APCI fragmentation patterns, it is possible to rapidly screen many more bacterial cultures using only small amounts of material (<50 mg) as well as environmental samples for these atypical structures and a rapidly growing suite of novel structures.

A review of tobacco BY-2 cells as an excellent system to study the synthesis and function of sterols and other isoprenoids.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. In this paper we review findings and observations made primarily with tobacco BY-2 cells (TBY-2), which have proven to be an excellent system in which to study the two biosynthetic pathways. A major advantage of these cells as an experimental system is their ability to readily take up specific inhibitors and stably- and/or radiolabeled precursors. This permits the functional elucidation of the role of isoprenoid end products and intermediates. Because TBY-2 cells undergo rapid cell division and can be synchronized within the cell cycle, they constitute a highly suitable test system for determination of those isoprenoids and intermediates that act as cell cycle inhibitors, thus giving an indication of which branches of the isoprenoid pathway are essential. Through chemical complementation; and use of precursors, intracellular compartmentation can be elucidated, as well as the extent to which the plastidial and cytosolic pathways contribute to the syntheses of specific groups of isoprenoids (e.g., sterols) via exchange of intermediates across membranes. These topics are discussed in the context of the pertinent literature.

Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate.

In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-(2)H]deoxy-d-xylulose ([4-(2)H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-(2)H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase.