4,071 research outputs found
Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems
The incorporation of [14C]mevalonate and [14C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system
Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq
BACKGROUND: Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. RESULTS: The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. CONCLUSION: This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots
Simvastatin inhibits TLR8 signaling in primary human monocytes and spontaneous TNF production from rheumatoid synovial membrane cultures
Simvastatin has been shown to have anti-inflammatory effects that are independent of its serum cholesterol lowering action, but the mechanisms by which these anti-inflammatory effects are mediated have not been elucidated. To explore the mechanism involved, the effect of simvastatin on Toll-like receptor (TLR) signalling in primary human monocytes was investigated. A short pre-treatment with simvastatin dose-dependently inhibited the production of tumor necrosis factor-α (TNF) in response to TLR8 (but not TLRs 2, 4, or 5) activation. Statins are known inhibitors of the cholesterol biosynthetic pathway, but intriguingly TLR8 inhibition could not be reversed by addition of mevalonate or geranylgeranyl pyrophosphate; downstream products of cholesterol biosynthesis. TLR8 signalling was examined in HEK 293 cells stably expressing TLR8, where simvastatin inhibited IKKα/β phosphorylation and subsequent NF-κB activation without affecting the pathway to AP-1. Since simvastatin has been reported to have anti-inflammatory effects in RA patients and TLR8 signalling contributes to TNF production in human RA synovial tissue in culture, simvastatin was tested in these cultures. Simvastatin significantly inhibited the spontaneous release of TNF in this model which was not reversed by mevalonate. Together, these results demonstrate a hitherto unrecognized mechanism of simvastatin inhibition of TLR8 signalling that may in part explain its beneficial anti-inflammatory effects
Phytol synthesis from geranylgeraniol in spinach chloroplasts
The reduction of /2-14C/-geranylgeranylpyrophosphate to phytylpyrophosphosphate is shown for the first time in chloroplasts. The esterification of exogenous /2-14C/-geranylgeranylpyrophosphate with endogenous chlorophyllide and the stepwise reduction of the pigment bound geranylgeraniol to phytol was also proved for spinach chloroplasts for the first time
Mutant-based model of two independent pathways for carotenoid-mediated chloroplast biogenesis in Arabidopsis embryos
Chloroplasts are essential for autonomous plant growth, and their biogenesis is a complex process requiring both plastid and nuclear genome. One of the essential factors required for chloroplast biogenesis are carotenoids. Carotenoids are synthesized in plastids, and it was shown that plastid localized methylerythritol 4-phosphate (MEP) pathway provides substrates for their biosynthesis. Here, we propose a model, using results of our own mutant analysis combined with the results of others, that a MEP-independent pathway, likely a mevalonate (MVA)-dependent pathway, provides intermediates for chloroplast biogenesis in Arabidopsis embryos. The pattern of this chloroplast biogenesis differs from the MEP-dependent chloroplast biogenesis. In MEP-dependent chloroplast biogenesis, chloroplasts are formed rather uniformly in the whole embryo, with stronger chlorophyll accumulation in cotyledons. In a MEP-independent pathway, chloroplasts are formed predominantly in the hypocotyl and in the embryonic root. We also show that this pattern of chlorophyll accumulation is common to MEP pathway mutants as well as to the mutant lacking geranylgeranyl diphosphate synthase 11 (GGPPS11) activity in plastids but expressing it in the cytosol (GGPPS11cyt). It was recently described that shorter GGPPS11 transcripts are present in Arabidopsis, and they can be translated into active cytosolic proteins. We therefore propose that the MEP-independent pathway for chloroplast biogenesis in Arabidopsis embryos is an MVA pathway that provides substrates for the synthesis of GGPP via GGPPS11cyt and this is then transported to plastids, where it is used for carotenoid biosynthesis and subsequently for chloroplast biogenesis mainly in the hypocotyl and in the embryonic root
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Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.
BACKGROUND:Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS:The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism
Constructing tailored isoprenoid products by structure-guided modification of geranylgeranyl reductase
The archaeal enzyme geranylgeranyl reductase (GGR) catalyzes hydrogenation of carbon-carbon double bonds to produce the saturated alkyl chains of the organism\u27s unusual isoprenoid-derived cell membrane. Enzymatic reduction of isoprenoid double bonds is of considerable interest both to natural products researchers and to synthetic biologists interested in the microbial production of isoprenoid drug or biofuel molecules. Here we present crystal structures of GGR from Sulfolobus acidocaldarius, including the structure of GGR bound to geranylgeranyl pyrophosphate (GGPP). The structures are presented alongside activity data that depict the sequential reduction of GGPP to H(6)GGPP via the intermediates H(2)GGPP and H(4)GGPP. We then modified the enzyme to generate sequence variants that display increased rates of H(6)GGPP production or are able to halt the extent of reduction at H(2)GGPP and H(4)GGPP. Crystal structures of these variants not only reveal the structural bases for their altered activities; they also shed light onto the catalytic mechanism employed
Cloning and characterisation of chlorophyll synthase from Avena sativa
The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence, of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in dark-grown and in light-grown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and N-phenylmaleimide (NPM) inhibit the enzyme activity. Site-directed mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wild-type and all other Cys-mutants with the exception of the mutant C304A are inhibited by N-phenylmaleimide, we conclude that the inhibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed
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