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The liverwort Frullania tamarisci (L.) Dumort produces large amounts of terpenoids, among others the sesquiterpene alcohol tamariscol. Tamariscol has an earthy woody fragrance, and the use in perfurmes and production of it was patented in 1984. The microbial terpene synthase-like (MTPSL) enzyme FtMTPSL6 is shown to be responsible for the biosynthesis of tamariscol. FtMTPSL6 was obtained through RNA sequencing of wild growing F. tamarisci along with six other MTPSLs (FtMTPSL1-7). The biochemical activity was determined for three of them, and two others where metabolic active, but the product could not be identified. The three characterized enzymes are the tamariscol synthase (FtMTPSL6), copaene synthase (FtMTPSL1) and gurjunene synthase (FtMTPSL3). Thus, the biosynthesis of the economically attractive compound tamariscol is established, and this opens up for further exploitation of this molecule., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To date, there is no functional characterization of EmGGPPS (from Elizabethkingia meningoseptica sp.F2) as enzymes catalyzing GGPP. In this research, maltose-binding protein (MBP), disulfide bond A (DbsA), disulfide bond C (DbsC), and two other small protein tags, GB1 (Protein G B1 domain) and ZZ (Protein A IgG ZZ repeat domain), were used as fusion partners to construct an EmGGPPS fusion expression system. The results indicated that the expression of MBP-EmGGPPS was higher than that of the other four fusion proteins in E. coli BL21 (DE3). Additionally, using EmGGPPS as a catalyst for the production of GGPP was verified using a color complementation assay in Escherichia coli. In parallel with it, the enzyme activity experiment in vitro showed that the EmGGPPS protein could produce GGPP, GPP and FPP. Finally, we successfully demonstrated MK-4 production in engineered E. coli by overexpression of EmGGPPS., (Copyright © 2021. Published by Elsevier Inc.)

A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastrene A and polytrichastrol A with new skeletons, besides known wanju-2,5-diene and thunbergol. The enzyme mechanism was investigated by isotopic labeling experiments and DFT calculations to explain an unusual ethyl group formation. Rationally designed exchanges of active site residues showed major functional switches, resulting for I66F in the production of five more new compounds, including polytrichastrene B and polytrichastrol B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju-2,5-diene., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Aims: Thymic carcinoma is a rare epithelial tumor, for which, optimal pharmacotherapeutic methods have not yet been established. To develop new drug treatments for thymic carcinoma, we investigated the effects of fluvastatin-mediated pharmacological inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) on thymic carcinoma., Main Methods: Thymic carcinoma tissue was surgically excised and HMGCR expression was assessed by immunohistochemistry. Ty82 human thymic carcinoma cells were treated with fluvastatin (1-10 μM) and their growth was monitored., Key Findings: HMGCR was expressed on carcinoma cells but not on normal epithelial cells in thymic tissue. Inhibition of HMGCR by fluvastatin suppressed cell proliferation and induced the death of Ty-82 human thymic carcinoma cells. Fluvastatin mediated its antitumor effects by blocking the production of geranylgeranyl-pyrophosphate (GGPP), an isoprenoid that is produced from mevalonate and binds to small GTPases, which promotes cell proliferation., Significance: Fluvastatin showed marked antitumor effects on thymic carcinoma. The results suggest that the statin has clinical benefits in thymic carcinoma management., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia 1 . Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications 2 . Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments 3 . However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids 4 , and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ 9 -tetrahydrocannabinolic acid, cannabidiolic acid, Δ 9 -tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway 5 . We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid 6 , as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases 7,8 . Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency 9 . We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Background: Following androgen deprivation for the treatment of advanced adenocarcinoma of the prostate, tumors can progress to neuroendocrine prostate cancer (NEPC). This transdifferentiation process is poorly understood, but trafficking of transcriptional factors and/or cytoskeletal rearrangements may be involved. We observed the role of geranylgeranylation in this process by treatment with digeranyl bisphosphonate (DGBP), a selective inhibitor of geranylgeranyl pyrophosphate synthase which blocks the prenylation of small GTPases such as Rho and Rab family proteins, including Cdc42 and Rac1., Methods: We examined the therapeutic potential of DGBP in LNCaP, C4-2B4, and 22Rv1 cell culture models. Cell morphology and protein expression were quantified to observe the development of the neuroendocrine phenotype in androgen-deprivation and abiraterone-treated LNCaP models of NEPC development. Luciferase reporter assays were utilized to examine AR activity, and immunofluorescence visualized the localization of AR within the cell., Results: Essential genes in the isoprenoid pathway, such as HMGCR, MVK, GGPS1, and GGT1, were highly expressed in a subset of castration resistant prostate cancers reported by Beltran et al. Under treatment with DGBP, nuclear localization of AR decreased in LNCaP, 22Rv1, and C4-2B4 cell lines, luciferase reporter activity was reduced in LNCaP and 22Rv1, and AR target gene transcription also decreased in LNCaP. Conversely, nuclear localization of AR was enhanced by the addition of GGOH. Finally, induction of the NEPC structural and molecular phenotype via androgen deprivation in LNCaP cells was inhibited by DGBP in a GGOH-dependent manner., Conclusions: DGBP is a novel compound with the potential to reduce AR transcriptional activity and inhibit PCa progression to NEPC phenotype. These results suggest that DGBP may be used to block cell growth and metastasis in both hormone therapy sensitive and resistant paradigms., (© 2018 Wiley Periodicals, Inc.)

The enzymatic synthesis of terpenes was investigated by using a cascade based on the mevalonic acid pathway. Suitable enzymes from all kingdoms of life were identified and combined to give rise to geosmin and patchoulol as representative compounds. The pathway was studied in three separate segments, which were subsequently combined in a ten-step cascade plus added cofactor regeneration systems. The cascade delivers farnesyl pyrophosphate with >40 % conversion and cyclises it to sesquiterpenes with >90 % conversion., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. In Escherichia coli , this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS). E. coli MG1655 exhibits wild-type morphology at all temperatures, but one of our laboratory strains (CS109) was highly aberrant when grown at 42°C. This strain contained mutations affecting the Und-P synthetic pathway genes uppS , ispH , and idi Normal morphology was restored by overexpressing uppS or by replacing the mutant ( uppS31 ) with the wild-type allele. Importantly, moving uppS31 into MG1655 was lethal even at 30°C, indicating that the altered enzyme was highly deleterious, but growth was restored by adding the CS109 versions of ispH and idi Purified UppS W31R was enzymatically defective at all temperatures, suggesting that it could not supply enough Und-P during rapid growth unless suppressor mutations were present. We conclude that cell wall synthesis is profoundly sensitive to changes in the pool of polyisoprenoids and that isoprenoid homeostasis exerts a particularly strong evolutionary pressure. IMPORTANCE Bacterial morphology is determined primarily by the overall structure of the semirigid macromolecule peptidoglycan. Not only does peptidoglycan contribute to cell shape, but it also protects cells against lysis caused by excess osmotic pressure. Because it is critical for bacterial survival, it is no surprise that many antibiotics target peptidoglycan biosynthesis. However, important gaps remain in our understanding about how this process is affected by peptidoglycan precursor availability. Here, we report that a mutation altering the enzyme that synthesizes Und-P prevents cells from growing at high temperatures and that compensatory mutations in enzymes functioning upstream of uppS can reverse this phenotype. The results highlight the importance of Und-P metabolism for maintaining normal cell wall synthesis and shape., (Copyright © 2018 American Society for Microbiology.)

Engineering photosynthetic bacteria to utilize a heterologous reaction center that contains a different (bacterio) chlorophyll could improve solar energy conversion efficiency by allowing cells to absorb a broader range of the solar spectrum. One promising candidate is the homodimeric type I reaction center from Heliobacterium modesticaldum. It is the simplest known reaction center and uses bacteriochlorophyll (BChl) g, which absorbs in the near-infrared region of the spectrum. Like the more common BChls a and b, BChl g is a true bacteriochlorin. It carries characteristic C3-vinyl and C8-ethylidene groups, the latter shared with BChl b. The purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides was chosen as the platform into which the engineered production of BChl g F , where F is farnesyl, was attempted. Using a strain of Rba. sphaeroides that produces BChl b P , where P is phytyl, rather than the native BChl a P , we deleted bchF, a gene that encodes an enzyme responsible for the hydration of the C3-vinyl group of a precursor of BChls. This led to the production of BChl g P . Next, the crtE gene was deleted, thereby producing BChl g carrying a THF (tetrahydrofarnesol) moiety. Additionally, the bchG Rs gene from Rba. sphaeroides was replaced with bchG Hm from Hba. modesticaldum. To prevent reduction of the tail, bchP was deleted, which yielded BChl g F . The construction of a strain producing BChl g F validates the biosynthetic pathway established for its synthesis and satisfies a precondition for assembling the simplest reaction center in a heterologous organism, namely the biosynthesis of its native pigment, BChl g F ., (Copyright © 2018. Published by Elsevier B.V.)

The mevalonate pathway has been described to play a key role in Alzheimer's disease (AD) physiopathology. Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are nonsterol isoprenoids derived from mevalonate, which serve as precursors to numerous human metabolites. They facilitate protein prenylation; hFPP and hGGPP synthases act as gateway enzymes to the prenylation of the small guanosine triphosphate (GTP)ase proteins such as RhoA and cdc42 that have been shown to facilitate phospho-tau (p-Tau, i.e., protein tau phosphorylated) production in the brain. In this study, a significant positive correlation was observed between the synthases mRNA prevalence and disease status (FPPS, p < 0.001, n = 123; GGPPS, p < 0.001, n = 122). The levels of mRNA for hFPPS and hGGPPS were found to significantly correlate with the amount of p-Tau protein levels (p < 0.05, n = 34) and neurofibrillary tangle density (p < 0.05, n = 39) in the frontal cortex. Interestingly, high levels of hFPPS and hGGPPS mRNA prevalence are associated with earlier age of onset in AD (p < 0.05, n = 58). Together, these results suggest that accumulation of p-Tau in the AD brain is related, at least in part, to increased levels of neuronal isoprenoids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5'- and 3'-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L -1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L -1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.

Bisphosphonates are widely used for the treatment of bone disorders. These drugs also inhibit the growth of a variety of protozoan parasites, such as Toxoplasma gondii , the etiologic agent of toxoplasmosis. The target of the most potent bisphosphonates is the isoprenoid biosynthesis pathway enzyme farnesyl diphosphate synthase (FPPS). Based on our previous work on the inhibitory effect of sulfur-containing linear bisphosphonates against T. gondii , we investigated the potential synergistic interaction between one of these derivatives, 1-[( n -heptylthio)ethyl]-1,1-bisphosphonate (C7S), and statins, which are potent inhibitors of the host 3-hydroxy-3-methyl glutaryl-coenzyme A reductase (3-HMG-CoA reductase). C7S showed high activity against the T. gondii bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS), which catalyzes the formation of FPP and GGPP (50% inhibitory concentration [IC 50 ] = 31 ± 0.01 nM [mean ± standard deviation]), and modest effect against the human FPPS (IC 50 = 1.3 ± 0.5 μM). We tested combinations of C7S with statins against the in vitro replication of T. gondii We also treated mice infected with a lethal dose of T. gondii with similar combinations. We found strong synergistic activities when using low doses of C7S, which were stronger in vivo than when tested in vitro We also investigated the synergism of several commercially available bisphosphonates with statins both in vitro and in vivo Our results provide evidence that it is possible to develop drug combinations that act synergistically by inhibiting host and parasite enzymes in vitro and in vivo ., (Copyright © 2017 American Society for Microbiology.)

Carotenoids are naturally occurring red, orange and yellow pigments that are synthesized by plants and some microorganisms and fulfill many important physiological functions. This chapter describes the distribution of carotenoid in microorganisms, including bacteria, archaea, microalgae, filamentous fungi and yeasts. We will also focus on their functional aspects and applications, such as their nutritional value, their benefits for human and animal health and their potential protection against free radicals. The central metabolic pathway leading to the synthesis of carotenoids is described as the three following principal steps: (i) the synthesis of isopentenyl pyrophosphate and the formation of dimethylallyl pyrophosphate, (ii) the synthesis of geranylgeranyl pyrophosphate and (iii) the synthesis of carotenoids per se, highlighting the differences that have been found in several carotenogenic organisms and providing an evolutionary perspective. Finally, as an example, the synthesis of the xanthophyll astaxanthin is discussed.

GGPPS catalyses the expression of GGPP, a key protein in the mevalonate metabolic pathway. HMG-CoA reductase inhibitor statins can induce liver injury by inhibiting GGPP. However, the function of GGPPS in liver injury has not yet been revealed. In this study, we found that GGPPS increased in liver injury and that GGPPS deletion augmented liver injury and fibrosis. GGPPS inhibition induced hepatocyte apoptosis, inflammation and TGF-β1 secretion, which activated hepatic stellate cells. Our findings imply that GGPPS deletion induces hepatocyte apoptosis, which makes the liver vulnerable to hepatotoxicity., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The sesquiterpene (+)-valencene is an aroma compound of citrus fruits and is used to flavor foods and drinks. Biosynthesis of (+)-valencene starts from farnesyl pyrophosphate, an intermediate of carotenoid biosynthesis. Corynebacterium glutamicum, the workhorse of the million-ton scale amino acid industry, is naturally pigmented as it synthesizes the rare fifty carbon atoms (C50) containing carotenoid decaprenoxanthin. Since the carotenoid pathway of this Gram-positive bacterium has previously been engineered for efficient production of several C50 and C40 carotenoids, its potential to produce a sesquiterpene was assessed. Growth of C. glutamicum was negatively affected by (+)-valencene, but overlaying n-dodecane as organic phase for extraction of (+)-valencene was shown to be biocompatible. Heterologous expression of the (+)-valencene synthase gene from the sweet orange Citrus sinensis was not sufficient to enable (+)-valencene production, likely because provision of farnesyl pyrophosphate (FPP) by endogenous prenyltransferases was too low. However, upon deletion of two endogenous prenyltransferase genes and heterologous expression of either FPP synthase gene ispA from Escherichia coli or ERG20 from Saccharomyces cerevisiae (+)-valence production by C. sinensis valencene synthase was observed. Employing the valencene synthase from Nootka cypress improved (+)-valencene titers 10 fold to 2.41±0.26mgl(-1) (+)-valencene, which is equivalent to 0.25±0.03mgg(-1) cell dry weight (CDW). This is the first report on sesquiterpene overproduction by recombinant C. glutamicum., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting. Paclitaxel (Taxol(®)) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11-27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

Background: Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7., Results: It was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation., Conclusions: This indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.

Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have undertaken an extensive survey of a group of epimerases originally named Gne, that were thought to be responsible for inter-conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc). The analysis builds on recent work clarifying the specificity of some of these epimerases. We find three well defined clades responsible for inter-conversion of the gluco- and galacto-configuration at C4 of different N-acetylhexosamines. Their major biological roles are the formation of UDP-GalNAc, UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) and undecaprenyl pyrophosphate-N-acetylgalactosamine (UndPP-GalNAc) from the corresponding glucose forms. We propose that the clade of UDP-GlcNAcA epimerase genes be named gnaB and the clade of UndPP-GlcNAc epimerase genes be named gnu, while the UDP-GlcNAc epimerase genes retain the name gne. The Gne epimerases, as now defined after exclusion of those to be named GnaB or Gnu, are in the same clade as the GalE 4-epimerases for inter-conversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). This work brings clarity to an area that had become quite confusing. The identification of distinct enzymes for epimerisation of UDP-GlcNAc, UDP-GlcNAcA and UndPP-GlcNAc will greatly facilitate allocation of gene function in polysaccharide gene clusters, including those found in bacterial genome sequences. A table of the accession numbers for the 295 proteins used in the analysis is provided to enable the major tree to be regenerated with the inclusion of additional proteins of interest. This and other suggestions for annotation of 4-epimerase genes will facilitate annotation.

Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Insulin-induced gene proteins (INSIGs) function in control of cellular cholesterol. Mammalian INSIGs exert control by directly interacting with proteins containing sterol-sensing domains (SSDs) when sterol levels are elevated. Mammalian 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR) undergoes sterol-dependent, endoplasmic-reticulum (ER)-associated degradation (ERAD) that is mediated by INSIG interaction with the HMGR SSD. The yeast HMGR isozyme Hmg2 also undergoes feedback-regulated ERAD in response to the early pathway-derived isoprene gernanylgeranyl pyrophosphate (GGPP). Hmg2 has an SSD, and its degradation is controlled by the INSIG homologue Nsg1. However, yeast Nsg1 promotes Hmg2 stabilization by inhibiting GGPP-stimulated ERAD. We have proposed that the seemingly disparate INSIG functions can be unified by viewing INSIGs as sterol-dependent chaperones of SSD clients. Accordingly, we tested the role of sterols in the Nsg1 regulation of Hmg2. We found that both Nsg1-mediated stabilization of Hmg2 and the Nsg1-Hmg2 interaction required the early sterol lanosterol. Lowering lanosterol in the cell allowed GGPP-stimulated Hmg2 ERAD. Thus, Hmg2-regulated degradation is controlled by a two-signal logic; GGPP promotes degradation, and lanosterol inhibits degradation. These data reveal that the sterol dependence of INSIG-client interaction has been preserved for over 1 billion years. We propose that the INSIGs are a class of sterol-dependent chaperones that bind to SSD clients, thus harnessing ER quality control in the homeostasis of sterols.

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co(2+) or Mn(2+) as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg(2+). In reaction with Co(2+), PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg(2+), PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co(2+), Mn(2+), or Mg(2+) in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.

Spontaneous intracranial hemorrhage is a debilitating form of stroke, often leading to death or permanent cognitive impairment. Many of the causative genes and the underlying mechanisms implicated in developmental cerebral-vascular malformations are unknown. Recent in vitro and in vivo studies in mice have shown inhibition of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway to be effective in stabilizing cranial vessels. Using a combination of pharmacological and genetic approaches to specifically inhibit the HMGCR pathway in zebrafish (Danio rerio), we demonstrate a requirement for this metabolic pathway in developmental vascular stability. Here we report that inhibition of HMGCR function perturbs cerebral-vascular stability, resulting in progressive dilation of blood vessels, followed by vessel rupture, mimicking cerebral cavernous malformation (CCM)-like lesions in humans and murine models. The hemorrhages in the brain are rescued by prior exogenous supplementation with geranylgeranyl pyrophosphate (GGPP), a 20-carbon metabolite of the HMGCR pathway, required for the membrane localization and activation of Rho GTPases. Consistent with this observation, morpholino-induced depletion of the β-subunit of geranylgeranyltransferase I (GGTase I), an enzyme that facilitates the post-translational transfer of the GGPP moiety to the C-terminus of Rho family of GTPases, mimics the cerebral hemorrhaging induced by the pharmacological and genetic ablation of HMGCR. In embryos with cerebral hemorrhage, the endothelial-specific expression of cdc42, a Rho GTPase involved in the regulation of vascular permeability, was significantly reduced. Taken together, our data reveal a metabolic contribution to the stabilization of nascent cranial vessels, requiring protein geranylgeranylation acting downstream of the HMGCR pathway., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Statins have demonstrated substantial benefits in supporting cardiovascular health. Older individuals are more likely to experience the well-known muscle-related side effects of statins compared with younger individuals. Elderly females may be especially vulnerable to statin-related muscle disorder. This review will collate and discuss statin-related muscular effects, examine their molecular and genetic basis, and how these apply specifically to elderly women. Developing strategies to reduce the incidence of statin-induced myopathy in older adult women could contribute to a significant reduction in the overall incidence of statin-induced muscle disorder in this vulnerable group of patients. Reducing statin-related muscle disorder would likely improve overall patient compliance, thereby leading to an increase in improved short- and long-term outcomes associated with appropriate use of statins.

A group of prenyltransferases produce linear lipids by catalyzing consecutive condensation reactions of farnesyl diphosphate (FPP) with specific numbers of isopentenyl diphosphate (IPP), a common building block of isoprenoid compounds. Depending on the stereochemistry of the double bonds formed during IPP condensation, these prenyltransferases are categorized as cis- and trans-types. Undecaprenyl diphosphate synthase (UPPS) that catalyzes chain elongation of FPP by consecutive condensation reactions with eight IPP, to form C₅₅ lipid carrier for bacterial cell wall biosynthesis, serves as a model for understanding cis-prenyltransferases. In this review, the current knowledge in UPPS kinetics, mechanisms, structures, and inhibitors is summarized.

In this case study, we designed a farnesyl pyrophosphate (FPP) biosynthetic network using hybrid functional Petri net with extension (HFPNe) which is derived from traditional Petri net theory and allows easy modeling with graphical approach of various types of entities in the networks together. Our main objective is to improve the production of FPP in yeast, which is further converted to amorphadiene (AD), a precursor of artemisinin (antimalarial drug). Natively, mevalonate (MEV) pathway is present in yeast. Methyl erythritol phosphate pathways (MEP) are present only in higher plant plastids and eubacteria, but not present in yeast. IPP and DAMP are common isomeric intermediate in these two pathways, which immediately yields FPP. By integrating these two pathways in yeast, we augmented the FPP synthesis approximately two folds higher (431.16 U/pt) than in MEV pathway alone (259.91 U/pt) by using HFPNe technique. Further enhanced FPP levels converted to AD by amorphadiene synthase gene yielding 436.5 U/pt of AD which approximately two folds higher compared to the AD (258.5 U/pt) synthesized by MEV pathway exclusively. Simulation and validation processes performed using these models are reliable with identified biological information and data.

Using reaction of moraprenyl phosphate with the known N-acetylsialyl chloride and the novel N,N-diacetylsialyl (Neu5Ac(2)) chloride α- and β-anomers of polyprenyl sialyl phosphate were synthesized for the first time. The α-selectivity dramatically increased when Neu5Ac(2) chloride was used as the glycosyl donor., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al. revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP. This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle. Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells., (© 2011 Landes Bioscience)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q(10) (CoQ(10)), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ(10) precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ(10) was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ(10)-producing E. coli strain resulted in an increase in CoQ(10) content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ(10) content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. The inhibition of this key enzyme in the mevalonate pathway leads to suppression of cell proliferation and induction of apoptosis. However, the molecular mechanism of apoptosis induction by statins is not well understood in glioblastoma. In the present study, we attempted to elucidate the mechanism by which statins induce apoptosis in C6 glioma cells., Methods: The cytotoxicity of statins toward the C6 glioma cells were evaluated using a cell viability assay. The enzyme activity of caspase-3 was determined using activity assay kits. The effects of statins on signal transduction molecules were determined by western blot analyses., Results: We found that statins inhibited cell proliferation and induced apoptosis in these cells. We also observed an increase in caspase-3 activity. The apoptosis induced by statins was not inhibited by the addition of farnesyl pyrophosphate, squalene, ubiquinone, and isopentenyladenine, but by geranylgeranyl-pyrophosphate (GGPP). Furthermore, statins decreased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt., Conclusions: These results suggest that statins induce apoptosis when GGPP biosynthesis is inhibited and consequently decreases the level of phosphorylated ERK1/2 and Akt. The results of this study also indicate that statins could be used as anticancer agents in glioblastoma.

Multiple studies have implicated the depletion of isoprenoid biosynthetic pathway intermediates in the induction of autophagy. However, the exact mechanism by which isoprenoid biosynthesis inhibitors induce autophagy has not been well established. We hypothesized that inhibition of farnesyl diphosphate synthase (FDPS) and geranylgeranyl diphosphate synthase (GGDPS) by bisphosphonates would induce autophagy by depleting cellular geranylgeranyl diphosphate (GGPP) and impairing protein geranylgeranylation. Herein, we show that an inhibitor of FDPS (zoledronate) and an inhibitor of GGDPS (digeranyl bisphosphonate, DGBP) induce autophagy in PC3 prostate cancer and MDA-MB-231 breast cancer cells as measured by accumulation of the autophagic marker LC3-II. Treatment of cells with lysosomal protease inhibitors [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) and pepstatin A] in combination with zoledronate or digeranyl bisphosphonate further enhances the formation of LC3-II, indicating that these compounds induce autophagic flux. It is noteworthy that the addition of exogenous GGPP prevented the accumulation of LC3-II and impairment of Rab6 (a GGTase II substrate) geranylgeranylation by isoprenoid pathway inhibitors (lovastatin, zoledronate, and DGBP). However, exogenous GGPP did not restore isoprenoid pathway inhibitor-induced impairment of Rap1a (a GGTase I substrate) geranylgeranylation. In addition, specific inhibitors of farnesyl transferase and geranylgeranyl transferase I are unable to induce autophagy in our system. Furthermore, the addition of bafilomycin A1 (an inhibitor of autophagy processing) enhanced the antiproliferative effects of digeranyl bisphosphonate. These results are the first to demonstrate that bisphosphonates induce autophagy. Our study suggests that induction of autophagy in PC3 cells with these agents is probably dependent upon impairment of geranylgeranylation of GGTase II substrates.

We present here a study of a eukaryotic trans-prenylsynthase from the malaria pathogen Plasmodium vivax. Based on the results of biochemical assays and contrary to previous indications, this enzyme catalyzes the production of geranylgeranyl pyrophosphate (GGPP) rather than farnesyl pyrophosphate (FPP). Structural analysis shows that the product length is constrained by a hydrophobic cavity formed primarily by a set of residues from the same subunit as the product as well as at least one other from the dimeric partner. Furthermore, Plasmodium GGPP synthase (GGPPS) can bind nitrogen-containing bisphosphonates (N-BPs) strongly with the energetically favorable cooperation of three Mg(2+), resulting in inhibition by this class of compounds at IC(50) concentrations below 100 nM. In contrast, human and yeast GGPPSs do not accommodate a third magnesium atom in the same manner, resulting in their insusceptibility to N-BPs. This differentiation is in part attributable to a deviation in a conserved motif known as the second aspartate-rich motif: whereas the aspartates at the start and end of the five-residue motif in FFPP synthases and P. vivax GGPPSs both participate in the coordination of the third Mg(2+), an asparagine is featured as the last residue in human and yeast GGPPSs, resulting in a different manner of interaction with nitrogen-containing ligands.

Natural terpenoids have elaborate structures and various bioactivities, making difficult their synthesis and labeling with isotopes. We report here the enzymatic total synthesis of plant hormone gibberellins (GAs) with recombinant biosynthetic enzymes from stable isotope-labeled acetate. Mevalonate (MVA) is a key intermediate for the terpenoid biosynthetic pathway. ¹³C-MVA was synthesized from ¹³C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from ¹³C-acetate and ¹³C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA₄ biosynthesis from ent-kaurene via GA₁₂ were prepared in yeast and Escherichia coli. All intermediates and the final product GA₄ were uniformly labeled with ¹³C without dilution by natural abundance when [U-¹³C₂] acetate was used. The ¹³C-NMR and MS data for [U-¹³C₂₀] ent-kaurene confirmed ¹³C-¹³C coupling, and no dilution with the ¹²C atom was observed.

The biosynthetic origin of isopentenyl diphosphate in the polyisoprenoid biosynthesis of the rubber-producing plant Eucommia ulmoides Oliver was elucidated for the first time by feeding experiments using 13C-labeled isotopomers of (RS)-mevalonate, 1-deoxy-D-xylulose-3,4,5-triacetate, 2C-methyl-D-erythritol-1,2,3,4-tetraacetate, and pyruvate. After 13C-labeled isotopomers were fed to the young seedlings, the polyisoprenoid fractions were prepared and analyzed by 13C NMR. The NMR data showed that the isoprene units of polyisoprenoid derived from isopentenyl diphosphate, which was biosynthesized using both mevalonate and 1-deoxy-D-xylulose-5-phosphate in E. ulmoides. It is assumed that the cross-talk of isopentenyl diphosphate, derived from both pathways, occurs during the biosynthesis of polyisoprenoid; therefore, it was observed in the formation of low-molecular weight isoprenoids.

Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene(s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism.

The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 A) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 A). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 alpha-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an alpha-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.

The WecA transferase is an integral membrane protein and a member of the polyprenyl phosphate N-acetylhexosamine-1-phosphate transferase superfamily. It initiates the biosynthesis of various bacterial cell envelope components such as the lipopolysaccharide O-antigen. We report on the first large-scale enzymatic synthesis, purification, and characterization of the undecaprenyl-pyrophosphoryl-N-acetylglucosamine product of the WecA transferase. This is an essential lipid intermediate for the biosynthesis of various bacterial cell envelope components. Its availability in a pure form will allow the biochemical and structural characterization of the various enzymes requiring it as a substrate for the synthesis of cell wall polymers.

Trichothecenes are isoprenoid mycotoxins produced in wheat infected with the filamentous fungus Fusarium graminearum. Some fungal genes for trichothecene biosynthesis (Tri genes) are known to be under control of transcription factors encoded by Tri6 and Tri10. Tri6 and Tri10 deletion mutants were constructed in order to discover additional genes regulated by these factors in planta. Both mutants were greatly reduced in pathogenicity and toxin production and these phenotypes were largely restored by genetic complementation with the wild-type gene. Transcript levels for over 200 genes were altered > or = twofold for Deltatri6 or Deltatri10 mutants including nearly all known Tri genes. Also reduced were transcript levels for enzymes in the isoprenoid biosynthetic pathway leading to farnesyl pyrophosphate, the immediate molecular precursor of trichothecenes. DNA sequences 5' to isoprenoid biosynthetic genes were enriched for the Tri6p DNA binding motif, YNAGGCC, in F. graminearum but not in related species that do not produce trichothecenes. To determine the effect of trichothecene metabolites on gene expression, cultures were treated with trichodiene, the first metabolic intermediate specific to the trichothecene biosynthetic pathway. A total of 153 genes were upregulated by added trichodiene and were significantly enriched for genes likely involved in cellular transport. Differentially regulated genes will be targeted for functional analysis to discover additional factors involved in toxin biosynthesis, toxin resistance and pathogenesis.

Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-beta-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-beta-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-beta-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-alpha-l-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-alpha-l-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.

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.

The trans-polyisoprene compounds are synthesized by trans-isoprenyl diphosphate synthase (IDS) with consecutive condensation of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP). The in vitro condensation by IDS does not proceed efficiently by hydrophobic interaction between IDS and the hydrocarbon of longer products. In the present study, the enzymatic synthesis of trans-polyisoprenyl diphosphates was attempted in an organic-aqueous dual-liquid phase system with thermostable enzymes obtained from Thermococcus kodakaraensis. The conversion from DMAPP to a longer-chain product was achieved in a dual-liquid phase system, and more than 80% of the products were recovered in the organic phase. When the mutant IDS-Y81S, in which Tyr81 is replaced with Ser, was used in the dual-phase system, productivity was enhanced about four times and the ratio of the longer-chain products was increased. Co-incubation of IPP isomerase from T. kodakaraensis with IDS or IDS-Y81S enabled the direct synthesis of polyisoprenyl diphosphates from IPPs.

Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, suppress cell proliferation and induce apoptosis in various cancer cell lines. However, the effects of statins in head and neck carcinoma have not been reported. In this study, we investigated the mechanism by which fluvastatin induces apoptosis in HSC-3 cells. An increase in caspase-3 activity was observed. The apoptosis induced by fluvastatin was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP) to the cell culture. When we examined the survival signals at the time of apoptotic induction, we also found that fluvastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Moreover, we also found that U0126, a MEK1/2 inhibitor, induces apoptosis in HSC-3 cells. These results suggested that fluvastatin induces apoptosis by inhibiting GGPP biosynthesis and consequently decreasing the level of phosphorylated ERK1/2. The results of this study also indicate that fluvastatin may be used as an anticancer agent for tongue carcinoma.

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. (32)P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo(2)-[4'-(32)P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.

The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a farnesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.

Acute inflammation in the lung is fundamentally important to host defence, but chronic or excessive inflammation leads to several common respiratory diseases, including asthma and acute respiratory distress syndrome. The resolution of inflammation is an active process. In health, events at the onset of acute inflammation establish biosynthetic circuits for specific chemical mediators that later serve as agonists to orchestrate a return to tissue homeostasis. In addition to an overabundance of pro-inflammatory stimuli, pathological inflammation can also result from defects in resolution signalling. The understanding of anti-inflammatory, pro-resolution molecules and their counter-regulatory signalling pathways is providing new insights into the molecular pathophysiology of lung disease and opportunities for the design of therapeutic strategies. In the present review, the growing family of lipid mediators of resolution is examined, including lipoxins, resolvins, protectins, cyclopentenones and presqualene diphosphate. Roles are uncovered for these compounds, or their structural analogues, in regulating airway inflammation.