The <Emphasis Type="Italic">pds2</Emphasis> mutation is a lesion in the <Emphasis Type="Italic">Arabidopsis</Emphasis> homogentisate solanesyltransferase gene involved in plastoquinone biosynthesis

Plastoquinone plays critical roles in photosynthesis, chlororespiration and carotenoid biosynthesis. The previously isolated pds2 mutant from Arabidopsis was deficient in tocopherol and plastoquinone accumulation, and the biochemical phenotype of this mutant could not be reversed by externally applied homogentisate, suggesting a later step in tocopherol and/or plastoquinone biosynthesis had been disrupted. Recently, the protein encoded by At3g11950 (AtHST) was shown to condense homogentisate with solanesyl diphosphate (SDP), the substrate for plastoquinone synthesis, but not phytyl diphosphate (PDP), the substrate for tocopherol biosynthesis. We have sequenced the AtHST allele in the pds2 mutant background and identified an in-frame 6 bp (2 aa) deletion in the gene. The pds2 mutation could be functionally complemented by constitutive expression of AtHST, demonstrating that the molecular basis for the pds2 mutation is this 6 bp-lesion in the AtHST gene. Confocal microscopy of EGFP tagged AtHST suggested that AtHST is localized to the chloroplast envelope, supporting the hypothesis that plastoquinone synthesis occurs in the plastid.     

Biosynthesis of tocopherols: A re-examination of the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol

A number of previous studies of the involvement of 2-methyl-6-phytyl-1,4-benzoquinol in the biosynthesis of α-tocopherol have failed to take account of the fact that this quinol and its quinone have very similar chromatographic properties to those of 2-methyl-3-phytyl-1,4-benzoquinol and 2-methyl-3-phytyl-1,4-benzoquinone respectively. It has now been shown that the two quinones can be separated from each other either by multidevelopment TLC or by HPLC and that the claims made earlier with regard to the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol in chloroplasts are correct. In particular, it has been established that this quinol is the only methyl phytylbenzoquinol formed from homogentisate and phytyl pyrophosphate in chloroplast preparations. It has also been shown for the first time that lettuce chloroplasts are able to synthesize ³H-labelled α- and γ-tocopherols from [methylene-³H] homogentisate.     

Unusual features of a recombinant apple alpha-farnesene synthase

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.     

Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG

In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle.     

Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Gö1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.     

Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity

We report on the cDNA cloning and characterization of a novel short-chain isoprenyl diphosphate synthase from the aphid Myzus persicae. Of the three IPPS cDNAs we cloned, two yielded prenyltransferase activity following expression in Escherichia coli; these cDNAs encode identical proteins except for the presence, in one of them, of an N-terminal mitochondrial targeting peptide. Although the aphid enzyme was predicted to be a farnesyl diphosphate synthase by BLASTP analysis, rMpIPPS, when isopentenyl diphosphate and dimethylallyl diphosphate are supplied as substrates, typically generated geranyl diphosphate (C10) as its main product, along with significant quantities of farnesyl diphosphate (C15). Analysis of an MpIPPS homology model pointed to substitutions that could confer GPP/FPP synthase activity to the aphid enzyme.     

Gene network reconstruction identifies the authentic trans-prenyl diphosphate synthase that makes the solanesyl moiety of ubiquinone-9 in Arabidopsis

Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed of a redox active benzoquinone ring attached to a prenyl side chain. The length of the latter varies among species, and depends upon the product specificity of a trans-long-chain prenyl diphosphate synthase that elongates an allylic diphosphate precursor. In Arabidopsis, this enzyme is assumed to correspond to an endoplasmic reticulum-located solanesyl diphosphate synthase, although direct genetic evidence was lacking. In this study, the reconstruction of the functional network of Arabidopsis genes linked to ubiquinone biosynthesis singled out an unsuspected solanesyl diphosphate synthase candidate--product of gene At2g34630--that, extraordinarily, had been shown previously to be targeted to plastids and to contribute to the biosynthesis of gibberellins. Green fluorescent protein (GFP) fusion experiments in tobacco and Arabidopsis, and complementation of a yeast coq1 knockout lacking mitochondrial hexaprenyl diphosphate synthase demonstrated that At2g34630 is also targeted to mitochondria. At2g34630 is the main--if not sole--contributor to solanesyl diphosphate synthase activity required for the biosynthesis of ubiquinone, as demonstrated by the dramatic (75-80%) reduction of the ubiquinone pool size in corresponding RNAi lines. Overexpression of At2g34630 gave up to a 40% increase in ubiquinone content compared to wild-type plants. None of the silenced or overexpressing lines, in contrast, displayed altered levels of plastoquinone. Phylogenetic analyses revealed that At2g34630 is the only Arabidopsis trans-long-chain prenyl diphosphate synthase that clusters with the Coq1 orthologs involved in the biosynthesis of ubiquinone in other eukaryotes.     

Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides

A highly purified and active cytochrome $\text{b}$-$\text{c}$₁ complex has been isolated from the chromatophores of the photosynthetic bacteria $\text{Rhodopseudomonas}\text{sphaeroides}$ R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome $\text{c}$ with a turnover number of 1500 per minute at 23° based on cytochrome $\text{c}$₁. It contains 8.3 nmoles of cytochromes $\text{b}$ and $\text{c}$₁ per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Characterisation of plant tocopherol cyclases and their overexpression in transgenic Brassica napus seeds

Tocopherols, collectively known as vitamin E, are only synthesised in photosynthetic organisms. Tocopherol cyclase (TC) catalyses the formation of the chromanol headgroup of the various tocopherol isoforms. TCs from Arabidopsis and maize (Zea mays) were expressed in Escherichia coli and purified. Analysis of the enzymatic properties revealed similarities but also differences between the two enzymes. Overexpression of chimeric TC gene constructs in developing seeds of transgenic rapeseed plants enhanced and modified the relative abundance of individual tocochromanol species in the seed oil, indicating a regulatory function of the enzyme in prenyllipid metabolism.     

Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.     

The first prenylation step in hyperforin biosynthesis

Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum (St. John's wort). Cell cultures of the related species Hypericum calycinum were found to contain a prenyltransferase activity which is likely to catalyze the first prenylation step in hyperforin biosynthesis. The enzyme was soluble and dependent on a divalent cation, with Fe2+ leading to maximum activity (Km=3.8 mM). The preferred prenyl donor was DMAPP (Km=0.46 mM) and the preferred prenyl acceptor was phlorisobutyrophenone (Km=0.52 mM). A broad pH optimum from 6.5 to 8.5 and a temperature optimum from 35 to 40 degrees C were observed. The formation of hyperforins in H. calycinum cell cultures was preceded by an increase in dimethylallyltransferase activity, with the maximum specific activity being 3.6 microkat/kg protein.     

Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.     

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and 1 was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed.     

Overexpression, purification, and characterization of selenomethionyl farnesyl diphosphate synthase of Bacillus stearothermophilus

To facilitate X-ray crystal structure solution of farnesyl diphosphate (FPP) synthase of Bacillus stearothermophilus, selenomethionyl recombinant enzyme was overproduced in a methionine (Met) auxotrophic strain of Escherichia coli, and purified to homogeneity by two chromatographic steps. About 50 mg of the pure selenomethionyl enzyme was obtained from 2 g of E. coli cells. Inductively coupled plasma (ICP) emission spectrometric analysis for selenium content showed that all of the Met residues in the FPP synthase were substituted by selenomethionine (SeMet). The selenomethionyl recombinant enzyme showed similar chromatographic behavior, heat stability, immunochemical property, product specificity, and kinetic parameters to those of the wild-type enzyme, indicating that SeMet substitution has little effect on the prenyltransferase with respect to substrate binding, enzymatic activity, and structure.     

Geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon. Cloning and characterization of a ket enzyme in shikonin biosynthesis.

Two cDNAs encoding geranyl diphosphate:4-hy- droxybenzoate 3-geranyltransferase were isolated from Lithospermum erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyl- transferases for ubiquinone biosynthesis. They were functionally expressed in yeast COQ2 disruptant and showed a strict substrate specificity for geranyl diphosphate as the prenyl donor, in contrast to ubiquinone biosynthetic enzymes, suggesting that they are involved in the biosynthesis of shikonin, a naphthoquinone secondary metabolite. Regulation of their expression by various culture conditions coincided with that of geranyltransferase activity and the secondary metabolites biosynthesized via this enzyme. This is the first established plant prenyltransferase that transfers the prenyl chain to an aromatic substrate.     

The structural basis of chain length control in Rv1086

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C₅₀) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.     

Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase

The L-galactose (Smirnoff-Wheeler) pathway represents the major route to L-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-L-galactose phosphorylases converting GDP-L-galactose to L-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of L-ascorbate. Here we report that the L-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the L-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-L-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and L-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the L-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells.     

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.