Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

PubMed

Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

1996-08-01

The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L

DOE PAGES

Thapa, Hem R.; Naik, Mandar T.; Okada, Shigeru; ...

2016-04-06

Here, the green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C 40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C 30 squalene. Confirming this hypothesis, the current study identifies C 20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encodingmore » a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C 15 and C 20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. In conclusion, this discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii.« less

A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice.

PubMed

Zhou, Fei; Wang, Cheng-Yuan; Gutensohn, Michael; Jiang, Ling; Zhang, Peng; Zhang, Dabing; Dudareva, Natalia; Lu, Shan

2017-06-27

In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits.

PubMed

Gutensohn, Michael; Orlova, Irina; Nguyen, Thuong T H; Davidovich-Rikanati, Rachel; Ferruzzi, Mario G; Sitrit, Yaron; Lewinsohn, Efraim; Pichersky, Eran; Dudareva, Natalia

2013-08-01

Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O. basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis1[OPEN

PubMed Central

Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E.; Chen, Ming; Zhou, Yongming; Yu, Bin

2015-01-01

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. PMID:26048882

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis.

PubMed

Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E; Chen, Ming; Zhou, Yongming; Yu, Bin; Cahoon, Edgar B

2015-08-01

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. © 2015 American Society of Plant Biologists. All Rights Reserved.

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

PubMed

Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

2015-11-20

The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production.

PubMed

Zhang, Chunyu; Cahoon, Rebecca E; Hunter, Sarah C; Chen, Ming; Han, Jixiang; Cahoon, Edgar B

2013-02-01

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT-independent pathway for tocotrienol synthesis has also been shown to occur by de-regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2-1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA-expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA-induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT-catalyzed tocotrienol synthesis. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

PubMed Central

Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

1998-01-01

Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae▿

PubMed Central

Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

2009-01-01

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

Toxoplasma gondii Relies on Both Host and Parasite Isoprenoids and Can Be Rendered Sensitive to Atorvastatin

PubMed Central

Li, Zhu-Hong; Ramakrishnan, Srinivasan; Striepen, Boris; Moreno, Silvia N. J.

2013-01-01

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites. PMID:24146616

Toxoplasma gondii relies on both host and parasite isoprenoids and can be rendered sensitive to atorvastatin.

PubMed

Li, Zhu-Hong; Ramakrishnan, Srinivasan; Striepen, Boris; Moreno, Silvia N J

2013-01-01

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.

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

PubMed

Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N; Bach, Thomas J

2009-01-01

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.

Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

PubMed

Misra, Rajesh Chandra; Garg, Anchal; Roy, Sudeep; Chanotiya, Chandan Singh; Vasudev, Prema G; Ghosh, Sumit

2015-11-01

Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases

PubMed Central

Keeling, Christopher I.; Chiu, Christine C.; Aw, Tidiane; Li, Maria; Henderson, Hannah; Tittiger, Claus; Weng, Hong-Biao; Blomquist, Gary J.; Bohlmann, Joerg

2013-01-01

The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon–carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors. PMID:24167290

Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis

PubMed Central

Collier, Christine; Griffin, Sholeem; Schuberth, Hans-Joachim; Sandra, Olivier; Smith, David G.; Mahan, Suman; Dieuzy-Labaye, Isabelle; Sheldon, I. Martin

2016-01-01

Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies. PMID:26673142

A Possible Trifunctional β-Carotene Synthase Gene Identified in the Draft Genome of Aurantiochytrium sp. Strain KH105

PubMed Central

Iwasaka, Hiroaki; Satoh, Ryota; Nagano, Akiko; Watanabe, Kenshi; Hisata, Kanako; Satoh, Noriyuki

2018-01-01

Labyrinthulomycetes have been regarded as a promising industrial source of xanthophylls, including astaxanthin and canthaxanthin, polyunsaturated fatty acids such as docosahexaenoic acid and docosapentaenoic acid, ω-3 oils, and terpenic hydrocarbons, such as sterols and squalene. A Thraustochytrid, Aurantiochytrium sp. KH105 produces carotenoids, including astaxanthin, with strong antioxidant activity. To gain genomic insights into this capacity, we decoded its 97-Mbp genome and characterized genes for enzymes involved in carotenoid biosynthesis. Interestingly, all carotenogenic genes, as well as other eukaryotic genes, appeared duplicated, suggesting that this strain is diploid. In addition, among the five genes involved in the pathway from geranylgeranyl pyrophosphate to astaxanthin, geranylgeranyl phytoene synthase (crtB), phytoene desaturase (crtI) and lycopene cyclase (crtY) were fused into single gene (crtIBY) with no internal stop codons. Functionality of the trifunctional enzyme, CrtIBY, to catalyze the reaction from geranylgeranyl diphosphate to β-carotene was confirmed using a yeast assay system and mass spectrometry. Furthermore, analyses of differential gene expression showed characteristic up-regulation of carotenoid biosynthetic genes during stationary and starvation phases under these culture conditions. This suggests genetic engineering events to promote more efficient production of carotenoids. We also showed an occurrence of crtIBY in other Thraustochytrid species. PMID:29642531

A Possible Trifunctional β-Carotene Synthase Gene Identified in the Draft Genome of Aurantiochytrium sp. Strain KH105.

PubMed

Iwasaka, Hiroaki; Koyanagi, Ryo; Satoh, Ryota; Nagano, Akiko; Watanabe, Kenshi; Hisata, Kanako; Satoh, Noriyuki; Aki, Tsunehiro

2018-04-09

Labyrinthulomycetes have been regarded as a promising industrial source of xanthophylls, including astaxanthin and canthaxanthin, polyunsaturated fatty acids such as docosahexaenoic acid and docosapentaenoic acid, ω-3 oils, and terpenic hydrocarbons, such as sterols and squalene. A Thraustochytrid, Aurantiochytrium sp. KH105 produces carotenoids, including astaxanthin, with strong antioxidant activity. To gain genomic insights into this capacity, we decoded its 97-Mbp genome and characterized genes for enzymes involved in carotenoid biosynthesis. Interestingly, all carotenogenic genes, as well as other eukaryotic genes, appeared duplicated, suggesting that this strain is diploid. In addition, among the five genes involved in the pathway from geranylgeranyl pyrophosphate to astaxanthin, geranylgeranyl phytoene synthase ( crtB ), phytoene desaturase ( crtI ) and lycopene cyclase ( crtY ) were fused into single gene ( crtIBY ) with no internal stop codons. Functionality of the trifunctional enzyme, CrtIBY, to catalyze the reaction from geranylgeranyl diphosphate to β-carotene was confirmed using a yeast assay system and mass spectrometry. Furthermore, analyses of differential gene expression showed characteristic up-regulation of carotenoid biosynthetic genes during stationary and starvation phases under these culture conditions. This suggests genetic engineering events to promote more efficient production of carotenoids. We also showed an occurrence of crtIBY in other Thraustochytrid species.

A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum

PubMed Central

Luo, Shi-Hong; Schmidt, Axel; Sun, Gui-Ling; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan

2016-01-01

Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-d-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms. PMID:26941091

Functional analysis of multiple carotenogenic genes from Lycium barbarum and Gentiana lutea L. for their effects on beta-carotene production in transgenic tobacco.

PubMed

Ji, Jing; Wang, Gang; Wang, Jiehua; Wang, Ping

2009-02-01

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, beta-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into beta-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and delta-carotene desaturase gene, lycopene beta-cyclase, lycopene epsilon-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced beta-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.

Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

PubMed

Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

2016-07-01

Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Control activity of yeast geranylgeranyl diphosphate synthase from dimer interface through H-bonds and hydrophobic interaction.

PubMed

Chang, Chih-Kang; Teng, Kuo-Hsun; Lin, Sheng-Wei; Chang, Tao-Hsin; Liang, Po-Huang

2013-04-23

Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased k(cat) values (200-250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS.

The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana

PubMed Central

Shibata, Yusuke; Ojika, Makoto; Sugiyama, Akifumi; Yazaki, Kazufumi; Jones, David A.; Kawakita, Kazuhito

2016-01-01

The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans. Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans. Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans. PMID:27102667

Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.

PubMed

Schilmiller, Anthony L; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L; Pichersky, Eran

2009-06-30

We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate

PubMed Central

Schilmiller, Anthony L.; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L.; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L.; Pichersky, Eran

2009-01-01

We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce β-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the “universal” substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes. PMID:19487664

Molecular cloning and nucleotide sequences of the genes for two essential proteins constituting a novel enzyme system for heptaprenyl diphosphate synthesis.

PubMed

Koike-Takeshita, A; Koyama, T; Obata, S; Ogura, K

1995-08-04

The genes encoding two dissociable components essential for Bacillus stearothermophilus heptaprenyl diphosphate synthase (all-trans-hexparenyl-diphosphate:isopentenyl-diphosphate hexaprenyl-trans-transferase, EC 2.5.1.30) were cloned, and their nucleotide sequences were determined. Sequence analyses revealed the presence of three open reading frames within 2,350 base pairs, designated as ORF-1, ORF-2, and ORF-3 in order of nucleotide sequence, which encode proteins of 220, 234, and 323 amino acids, respectively. Deletion experiments have shown that expression of the enzymatic activity requires the presence of ORF-1 and ORF-3, but ORF-2 is not essential. As a result, this enzyme was proved genetically to consist of two different protein compounds with molecular masses of 25 kDa (Component I) and 36 kDa (Component II), encoded by two of the three tandem genes. The protein encoded by ORF-1 has no similarity to any protein so far registered. However, the protein encoded by ORF-3 shows a 32% similarity to the farnesyl diphosphate synthase of the same bacterium and has seven highly conserved regions that have been shown typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J. Biochem. (Tokyo) 113, 355-363).

Structure of the ent -Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudolf, Jeffrey D.; Dong, Liao-Bin; Cao, Hongnan

Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three alpha-helical domains (alpha beta gamma), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (alpha) and type II TSs (beta gamma). Type II DTSs of bacterialmore » origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtnaT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 angstrom, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg2+-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.« less

Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase

PubMed Central

2016-01-01

Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg2+-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs. PMID:27490479

Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase.

PubMed

Rudolf, Jeffrey D; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2016-08-31

Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate.

PubMed

Gutensohn, Michael; Nguyen, Thuong T H; McMahon, Richard D; Kaplan, Ian; Pichersky, Eran; Dudareva, Natalia

2014-07-01

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

1999-01-01

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

1999-03-02

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

Insights Into the Bifunctional Aphidicolan-16-ß-ol Synthase Through Rapid Biomolecular Modeling Approaches.

PubMed

Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B

2018-01-01

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of the enzyme's active site and that the geranylgeranyl diphosphate derived pyrophosphate moiety remains in the ACS active site thereby directing the cyclization process. Our cumulative data confirm that amino acids constituting the G-loop of diterpene synthases are involved in the open to the closed, catalytically active enzyme conformation. This study demonstrates that a simple and rapid biomolecular modeling procedure can predict catalytically relevant amino acids. The approach reduces computational and experimental screening efforts for diterpene synthase structure-function analyses.

Insights into the bifunctional Aphidicolan-16-ß-ol synthase through rapid biomolecular modelling approaches

NASA Astrophysics Data System (ADS)

Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B.

2018-04-01

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modelling techniques offer an alternative route to study the enzyme’s reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modelling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modelling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789 and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modelling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of the enzyme’s active site and that the geranylgeranyl diphosphate derived pyrophosphate moiety remains in the ACS active site thereby directing the cyclization process. Our cumulative data confirm that amino acids constituting the G-loop of diterpene synthases are involved in the open to the closed, catalytically active enzyme conformation. This study demonstrates that a simple and rapid biomolecular modelling procedure can predict catalytically relevant amino acids. The approach reduces computational and experimental screening efforts for diterpene synthase structure-function analyses.

Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms1[W][OA

PubMed Central

Keeling, Christopher I.; Dullat, Harpreet K.; Yuen, Mack; Ralph, Steven G.; Jancsik, Sharon; Bohlmann, Jörg

2010-01-01

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms. PMID:20044448

Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon.

PubMed

Toyomasu, Tomonobu; Miyamoto, Koji; Shenton, Matthew R; Sakai, Arisa; Sugawara, Chizu; Horie, Kiyotaka; Kawaide, Hiroshi; Hasegawa, Morifumi; Chuba, Masaru; Mitsuhashi, Wataru; Yamane, Hisakazu; Kurata, Nori; Okada, Kazunori

2016-11-18

Cultivated rice (Oryza sativa) possesses various labdane-related diterpene synthase genes, homologs of ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins. Copyright © 2016 Elsevier Inc. All rights reserved.

Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection.

PubMed

Wang, Xiu-Xing; Ying, Pu; Diao, Fan; Wang, Qiang; Ye, Dan; Jiang, Chen; Shen, Ning; Xu, Na; Chen, Wei-Bo; Lai, Shan-Shan; Jiang, Shan; Miao, Xiao-Li; Feng, Jin; Tao, Wei-Wei; Zhao, Ning-Wei; Yao, Bing; Xu, Zhi-Peng; Sun, Hai-Xiang; Li, Jian-Min; Sha, Jia-Hao; Huang, Xing-Xu; Shi, Qing-Hua; Tang, Hong; Gao, Xiang; Li, Chao-Jun

2013-07-29

Mumps commonly affects children 5-9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell-only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients' fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-κB signaling pathways were constitutively activated in GGPPS(-/-) Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS(-/-) Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility.

Bacterial Diterpene Synthases: New Opportunities for Mechanistic Enzymology and Engineered Biosynthesis

PubMed Central

Smanski, Michael J.; Peterson, Ryan M.; Huang, Sheng-Xiong; Shen, Ben

2012-01-01

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering. PMID:22445175

Expression of lima bean terpene synthases in rice enhances recruitment of a beneficial enemy of a major rice pest.

PubMed

Li, Fengqi; Li, Wei; Lin, Yong-Jun; Pickett, John A; Birkett, Michael A; Wu, Kongming; Wang, Guirong; Zhou, Jing-Jiang

2018-01-01

Volatile terpenoids play a key role in plant defence against herbivory by attracting parasitic wasps. We identified seven terpene synthase genes from lima bean, Phaseolus lunatus L. following treatment with either the elicitor alamethicin or spider mites, Tetranychus cinnabarinus. Four of the genes (Pltps2, Pltps3, Pltps4 and Pltps5) were up-regulated with their derived proteins phylogenetically clustered in the TPS-g subfamily and PlTPS3 positioned at the base of this cluster. Recombinant PlTPS3 was able to convert geranyl diphosphate and farnesyl diphosphate to linalool and (E)-nerolidol, the latter being precursor of the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Recombinant PlTPS4 showed a different substrate specificity and produced linalool and (E)-nerolidol, as well as (E,E)-geranyllinalool from geranylgeranyl diphosphate. Transgenic rice expressing Pltps3 emitted significantly more (S)-linalool and DMNT than wild-type plants, whereas transgenic rice expressing Pltps4 produced (S)-linalool, DMNT and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT). In laboratory bioassays, female Cotesia chilonis, the natural enemy of the striped rice stemborer, Chilo suppressalis, were significantly attracted to the transgenic plants and their volatiles. We further confirmed this with synthetic blends mimicking natural rice volatile composition. Our study demonstrates that the transformation of rice to produce volatile terpenoids has the potential to enhance plant indirect defence through natural enemy recruitment. © 2017 John Wiley & Sons Ltd.

Genome-Wide Identification of Differentially Expressed Genes Associated with the High Yielding of Oleoresin in Secondary Xylem of Masson Pine (Pinus massoniana Lamb) by Transcriptomic Analysis

PubMed Central

Liu, Qinghua; Zhou, Zhichun; Wei, Yongcheng; Shen, Danyu; Feng, Zhongping; Hong, Shanping

2015-01-01

Masson pine is an important timber and resource for oleoresin in South China. Increasing yield of oleoresin in stems can raise economic benefits and enhance the resistance to bark beetles. However, the genetic mechanisms for regulating the yield of oleoresin were still unknown. Here, high-throughput sequencing technology was used to investigate the transcriptome and compare the gene expression profiles of high and low oleoresin-yielding genotypes. A total of 40,690,540 reads were obtained and assembled into 137,499 transcripts from the secondary xylem tissues. We identified 84,842 candidate unigenes based on sequence annotation using various databases and 96 unigenes were candidates for terpenoid backbone biosynthesis in pine. By comparing the expression profiles of high and low oleoresin-yielding genotypes, 649 differentially expressed genes (DEGs) were identified. GO enrichment analysis of DEGs revealed that multiple pathways were related to high yield of oleoresin. Nine candidate genes were validated by QPCR analysis. Among them, the candidate genes encoding geranylgeranyl diphosphate synthase (GGPS) and (-)-alpha/beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype, while tricyclene synthase revealed lower expression level, which was in good agreement with the GC/MS result. In addition, DEG encoding ABC transporters, pathogenesis-related proteins (PR5 and PR9), phosphomethylpyrimidine synthase, non-specific lipid-transfer protein-like protein and ethylene responsive transcription factors (ERFs) were also confirmed to be critical for the biosynthesis of oleoresin. The next-generation sequencing strategy used in this study has proven to be a powerful means for analyzing transcriptome variation related to the yield of oleoresin in masson pine. The candidate genes encoding GGPS, (-)-alpha/beta-pinene, tricyclene synthase, ABC transporters, non-specific lipid-transfer protein-like protein, phosphomethylpyrimidine synthase, ERFs and pathogen responses may play important roles in regulating the yield of oleoresin. These DEGs are worthy of special attention in future studies. PMID:26167875

Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga Botryococcus braunii Race L.

PubMed

Thapa, Hem R; Tang, Su; Sacchettini, James C; Devarenne, Timothy P

2017-09-15

Recently, the biosynthetic pathway for lycopadiene, a C 40 tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C 20 geranylgeranyl diphosphate (GGPP) molecules to produce C 40 lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C 15 farnesyl diphosphate (FPP) and C 20 phytyl diphosphate in addition to GGPP as substrates. These three substrates can be combined by LOS individually or in combinations to produce six different hydrocarbons of C 30 , C 35 , and C 40 chain lengths. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignment with several SS proteins as well as a structural comparison with the human SS (HSS) crystal structure. Characterization of the LOS mutants in vitro identified Ser276 and Ala288 in the LOS active site as key amino acids responsible for controlling substrate binding, and thus the promiscuity of this enzyme. Mutating these residues to those found in HSS largely converted LOS from lycopaoctaene production to C 30 squalene production. Furthermore, these studies were confirmed in vivo by expressing LOS in E. coli cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length.

Monoterpene synthases from common sage (Salvia officinalis)

DOEpatents

Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

1999-01-01

cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

PubMed

Garg, Anchal; Agrawal, Lalit; Misra, Rajesh Chandra; Sharma, Shubha; Ghosh, Sumit

2015-09-02

Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs. De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs. Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

Synergistic Substrate Inhibition of ent-Copalyl Diphosphate Synthase: A Potential Feed-Forward Inhibition Mechanism Limiting Gibberellin Metabolism1[OA

PubMed Central

Prisic, Sladjana; Peters, Reuben J.

2007-01-01

Gibberellins (GAs) or gibberellic acids are ubiquitous diterpenoid phytohormones required for many aspects of plant growth and development, including repression of photosynthetic pigment production (i.e. deetiolation) in the absence of light. The committed step in GA biosynthesis is catalyzed in plastids by ent-copalyl diphosphate synthase (CPS), whose substrate, (E,E,E,)-geranylgeranyl diphosphate (GGPP), is also a direct precursor of carotenoids and the phytol side chain of chlorophyll. Accordingly, during deetiolation, GA production is repressed, whereas flux toward these photosynthetic pigments through their common GGPP precursor is dramatically increased. How this is accomplished has been unclear because no mechanism for regulation of CPS activity has been reported. We present here kinetic analysis of recombinant pseudomature CPS from Arabidopsis (Arabidopsis thaliana; rAtCPS) demonstrating that Mg2+ and GGPP exert synergistic substrate inhibition effects on CPS activity. These results suggest that GA metabolism may be limited by feed-forward inhibition of CPS; in particular, the effect of Mg2+ because light induces increases in plastid Mg2+ levels over a similar range as that observed here to affect rAtCPS activity. Notably, this effect is most pronounced in the GA-specific AtCPS because the corresponding activity of the resin acid biosynthetic enzyme abietadiene synthase is 100-fold less sensitive to [Mg2+]. Furthermore, Mg2+ allosterically activates the plant porphobilinogen synthase involved in chlorophyll production. Hence, Mg2+ may have a broad role in regulating plastidial metabolic flux during deetiolation. Finally, the observed synergistic substrate/feed-forward inhibition of CPS also seems to provide a novel example of direct regulation of enzymatic activity in hormone biosynthesis. PMID:17384166

A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro[S

PubMed Central

Wasko, Brian M.; Smits, Jacqueline P.; Shull, Larry W.; Wiemer, David F.; Hohl, Raymond J.

2011-01-01

Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion. PMID:21903868

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil, Anthonomus grandis.

PubMed

Taban, A Huma; Tittiger, Claus; Blomquist, Gary J; Welch, William H

2009-06-01

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full-length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three-dimensional structure of coleopteran FPPS was determined and compared to the X-ray crystal structure of avian FPPS. The alpha-helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. (c) 2009 Wiley Periodicals, Inc.

Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

PubMed

Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

2017-08-26

Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

Physical Mapping of bchG, orf427, and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides: Functional Assignment of the Bacteriochlorophyll Synthetase Gene

PubMed Central

Addlesee, Hugh A.; Fiedor, Leszek; Hunter, C. Neil

2000-01-01

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248–7255, 1999). An operonal arrangement of the genes is proposed. PMID:10809697

Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses

PubMed Central

2012-01-01

Background Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3) that have been widely characterized in rice, maize and sorghum. Results In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress. Conclusions We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses. PMID:22672222

A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase from Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Byres, Emma; Martin, David M. A.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk

2005-06-01

The gene encoding the putative mevalonate diphosphate decarboxylase, an enzyme from the mevalonate pathway of isoprenoid precursor biosynthesis, has been cloned from T. brucei. Recombinant protein has been expressed, purified and highly ordered crystals obtained and characterized to aid the structure–function analysis of this enzyme. Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in spacemore » group P2{sub 1}, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient V{sub M} of 2.5 Å{sup 3} Da{sup −1} corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.« less

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

PubMed

Yazaki, Kazufumi; Kunihisa, Miyuki; Fujisaki, Takahiro; Sato, Fumihiko

2002-02-22

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 biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

PubMed

Demissie, Zerihun A; Erland, Lauren A E; Rheault, Mark R; Mahmoud, Soheil S

2013-03-01

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae.

PubMed

Dai, Zhubo; Liu, Yi; Huang, Luqi; Zhang, Xueli

2012-11-01

Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well-known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial over-expression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L. Finally, fed-batch fermentation was performed, and 488 mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. Copyright © 2012 Wiley Periodicals, Inc.

The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

PubMed

Wu, Feng-Li; Shi, Liang; Yao, Jian; Ren, Ang; Zhou, Chao; Mu, Da-Shuai; Zhao, Ming-Wen

2013-01-01

An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of β-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

A homomeric geranyl diphosphate synthase-encoding gene from Camptotheca acuminata and its combinatorial optimization for production of geraniol in Escherichia coli.

PubMed

Yang, Lixia; Jiang, Liangzhen; Li, Wei; Yang, Yun; Zhang, Guolin; Luo, Yinggang

2017-10-01

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.

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

PubMed

Kumar, Hitesh; Kumar, Sanjay

2013-09-15

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia. Copyright © 2013 Elsevier B.V. All rights reserved.

Medically important carotenoids from Momordica charantia and their gene expressions in different organs.

PubMed

Cuong, Do Manh; Arasu, Mariadhas Valan; Jeon, Jin; Park, Yun Ji; Kwon, Soon-Jae; Al-Dhabi, Naif Abdullah; Park, Sang Un

2017-12-01

Carotenoids, found in the fruit and different organs of bitter melon ( Momordica charantia ), have attracted great attention for their potential health benefits in treating several major chronic diseases. Therefore, study related to the identification and quantification of the medically important carotenoid metabolites is highly important for the treatment of various disorderes. The present study involved in the identification and quantification of the various carotenoids present in the different organs of M. charantia and the identification of the genes responsible for the accumulation of the carotenoids with respect to the transcriptome levels were investigated. In this study, using the transcriptome database of bitter melon, a partial-length cDNA clone encoding geranylgeranyl pyrophosphate synthase ( McGGPPS2 ), and several full-length cDNA clones encoding geranylgeranyl pyrophosphate synthase ( McGGPPS1 ), zeta-carotene desaturase ( McZDS ), lycopene beta-cyclase ( McLCYB ), lycopene epsilon cyclases ( McLCYE1 and McLCYE2 ), beta-carotene hydroxylase ( McCHXB ), and zeaxanthin epoxidase ( McZEP ) were identified in bitter melon . The expression levels of the mRNAs encoding these eight putative biosynthetic enzymes, as well as the accumulation of lycopene, α-carotene, lutein, 13Z-β-carotene, E-β-carotene, 9Z-β-carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin, and violaxanthin were investigated in different organs from M. charantia as well as in the four different stages of its fruit maturation. Transcripts were found to be constitutively expressed at high levels in the leaves where carotenoids were also found at the highest levels . Collectively, these results indicate that the putative McGGPPS2, McZDS, McLCYB, McLCYE1, McLCYE2, and McCHXB enzymes might be key factors in controlling carotenoid content in bitter melon . In conclusion, the over expression of the carotenoid biosynthetic genes from M. charantia crops to increase the yield of these medically important carotenoids.

Dynamic control of ERG20 expression combined with minimized endogenous downstream metabolism contributes to the improvement of geraniol production in Saccharomyces cerevisiae.

PubMed

Zhao, Jianzhi; Li, Chen; Zhang, Yan; Shen, Yu; Hou, Jin; Bao, Xiaoming

2017-01-31

Microbial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes. Geraniol, a valuable monoterpene alcohol, is widely used in cosmetic, perfume, pharmaceutical and it is also a potential gasoline alternative. Previously, we constructed a geraniol production strain by engineering the mevalonate pathway together with the expression of a high-activity geraniol synthase. In this study, we further improved the geraniol production through reducing the endogenous metabolism of geraniol and controlling the precursor geranyl diphosphate flux distribution. The deletion of OYE2 (encoding an NADPH oxidoreductase) or ATF1 (encoding an alcohol acetyltransferase) both involving endogenous conversion of geraniol to other terpenoids, improved geraniol production by 1.7-fold or 1.6-fold in batch fermentation, respectively. In addition, we found that direct down-regulation of ERG20 expression, the branch point regulating geranyl diphosphate flux, does not improve geraniol production. Therefore, we explored dynamic control of ERG20 expression to redistribute the precursor geranyl diphosphate flux and achieved a 3.4-fold increase in geraniol production after optimizing carbon source feeding. Furthermore, the combination of dynamic control of ERG20 expression and OYE2 deletion in LEU2 prototrophic strain increased geraniol production up to 1.69 g/L with pure ethanol feeding in fed-batch fermentation, which is the highest reported production in engineered yeast. An efficient geraniol production platform was established by reducing the endogenous metabolism of geraniol and by controlling the flux distribution of the precursor geranyl diphosphate. The present work also provides a production basis to synthesis geraniol-derived chemicals, such as monoterpene indole alkaloids.

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

PubMed Central

2012-01-01

Background Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes. PMID:22834731

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture.

PubMed

Caniard, Anne; Zerbe, Philipp; Legrand, Sylvain; Cohade, Allison; Valot, Nadine; Magnard, Jean-Louis; Bohlmann, Jörg; Legendre, Laurent

2012-07-26

Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases*

PubMed Central

Jones, Christopher G.; Moniodis, Jessie; Zulak, Katherine G.; Scaffidi, Adrian; Plummer, Julie A.; Ghisalberti, Emilio L.; Barbour, Elizabeth L.; Bohlmann, Jörg

2011-01-01

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. PMID:21454632

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula

PubMed Central

Demissie, Zerihun A.; Erland, Lauren A. E.; Rheault, Mark R.; Mahmoud, Soheil S.

2013-01-01

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s−1, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering. PMID:23306202

Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli.

PubMed

Chen, Fei; Li, Wei; Jiang, Liangzhen; Pu, Xiang; Yang, Yun; Zhang, Guolin; Luo, Yinggang

2016-09-01

Geraniol synthase (GES) catalyzes the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries. Here we report the functional characterization of CaGES from Camptotheca acuminata, a camptothecin-producing plant, and its application in production of geraniol in Escherichia coli. The full-length cDNA of CaGES was obtained from overlap extension PCR amplification. The intact and N-terminus-truncated CaGESs were overexpressed in E. coli and purified to homogeneity. Recombinant CaGES showed the conversion activity from GPP to geraniol. To produce geraniol in E. coli using tCaGES, the biosynthetic precursor GPP should be supplied and transferred to the catalytic pocket of tCaGES. Thus, ispA(S80F), a mutant of farnesyl diphosphate (FPP) synthase, was prepared to produce GPP via the head-to-tail condensation of isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A slight increase of geraniol production was observed in the fermentation broth of the recombinant E. coli harboring tCaGES and ispA(S80F). To enhance the supply of IPP and DMAPP, the encoding genes involved in the whole mevalonic acid biosynthetic pathway were introduced to the E. coli harboring tCaGES and the ispA(S80F) and a significant increase of geraniol yield was observed. The geraniol production was enhanced to 5.85 ± 0.46 mg L(-1) when another copy of ispA(S80F) was introduced to the above recombinant strain. The following optimization of medium composition, fermentation time, and addition of metal ions led to the geraniol production of 48.5 ± 0.9 mg L(-1). The present study will be helpful to uncover the biosynthetic enigma of camptothecin and tCaGES will be an alternative to selectively produce geraniol in E. coli with other metabolic engineering approaches.

Blue-light irradiation up-regulates the ent-kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens.

PubMed

Miyazaki, Sho; Toyoshima, Hikaru; Natsume, Masahiro; Nakajima, Masatoshi; Kawaide, Hiroshi

2014-07-01

We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth. Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurenoic acid via ent-kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent-kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent-kaurene and ent-kaurenoic acid. This indicates that derivatives of ent-kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent-kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent-kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent-kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.

Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis.

PubMed

Rai, Avanish; Smita, Shachi S; Singh, Anup Kumar; Shanker, Karuna; Nagegowda, Dinesh A

2013-09-01

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.

Leigh Syndrome with Nephropathy and CoQ10 Deficiency Due to decaprenyl diphosphate synthase subunit 2 (PDSS2) Mutations

PubMed Central

López, Luis Carlos ; Schuelke, Markus ; Quinzii, Catarina M. ; Kanki, Tomotake ; Rodenburg, Richard J. T. ; Naini, Ali ; DiMauro, Salvatore ; Hirano, Michio 

2006-01-01

Coenzyme Q10 (CoQ10) is a vital lipophilic molecule that transfers electrons from mitochondrial respiratory chain complexes I and II to complex III. Deficiency of CoQ10 has been associated with diverse clinical phenotypes, but, in most patients, the molecular cause is unknown. The first defect in a CoQ10 biosynthetic gene, COQ2, was identified in a child with encephalomyopathy and nephrotic syndrome and in a younger sibling with only nephropathy. Here, we describe an infant with severe Leigh syndrome, nephrotic syndrome, and CoQ10 deficiency in muscle and fibroblasts and compound heterozygous mutations in the PDSS2 gene, which encodes a subunit of decaprenyl diphosphate synthase, the first enzyme of the CoQ10 biosynthetic pathway. Biochemical assays with radiolabeled substrates indicated a severe defect in decaprenyl diphosphate synthase in the patient’s fibroblasts. This is the first description of pathogenic mutations in PDSS2 and confirms the molecular and clinical heterogeneity of primary CoQ10 deficiency. PMID:17186472

Analysis of the enzymatic formation of citral in the glands of sweet basil.

PubMed

Iijima, Yoko; Wang, Guodong; Fridman, Eyal; Pichersky, Eran

2006-04-15

Basil glands of the Sweet Dani cultivar contain high levels of citral, a mixture of geranial and its cis-isomer neral, as well as low levels of geraniol and nerol. We have previously reported the identification of a cDNA from Sweet Dani that encodes an enzyme responsible for the formation of geraniol from geranyl diphosphate in the glands, and that these glands cannot synthesize nerol directly from geranyl diphosphate. Here, we report the identification of two basil cDNAs encoding NADP+-dependent dehydrogenases that can use geraniol as the substrate. One cDNA, designated CAD1, represents a gene whose expression is highly specific to gland cells of all three basil cultivars examined, regardless of their citral content, and encodes an enzyme with high sequence similarity to known cinnamyl alcohol dehydrogenases (CADs). The enzyme encoded by CAD1 reversibly oxidizes geraniol to produce geranial (which reversibly isomerizes to neral via keto-enol tautomerization) at half the efficiency compared with its activity with cinnamyl alcohol. CAD1 does not use nerol and neral as substrates. A second cDNA, designated GEDH1, encodes an enzyme with sequence similarity to CAD1 that is capable of reversibly oxidizing geraniol and nerol in equal efficiency, and prolonged incubation of geraniol with GEDH1 in vitro produces not only geranial and neral, but also nerol. GEDH1 is also active, although at a lower efficiency, with cinnamyl alcohol. However, GEDH1 is expressed at low levels in glands of all cultivars compared with its expression in leaves. These and additional data presented indicate that basil glands may contain additional dehydrogenases capable of oxidizing geraniol.

Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product.

PubMed Central

Finegold, A A; Johnson, D I; Farnsworth, C C; Gelb, M H; Judd, S R; Glomset, J A; Tamanoi, F

1991-01-01

Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated. Images PMID:2034682

Arabidopsis Chlorophyll Biosynthesis: An Essential Balance between the Methylerythritol Phosphate and Tetrapyrrole Pathways[C][W

PubMed Central

Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

2013-01-01

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)–derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis. PMID:24363312

Arabidopsis chlorophyll biosynthesis: an essential balance between the methylerythritol phosphate and tetrapyrrole pathways.

PubMed

Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

2013-12-01

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)-derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis.

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

PubMed

Jones, Christopher G; Moniodis, Jessie; Zulak, Katherine G; Scaffidi, Adrian; Plummer, Julie A; Ghisalberti, Emilio L; Barbour, Elizabeth L; Bohlmann, Jörg

2011-05-20

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Directed evolution of polymerase function by compartmentalized self-replication.

PubMed

Ghadessy, F J; Ong, J L; Holliger, P

2001-04-10

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

The Tomato Terpene Synthase Gene Family1[W][OA

PubMed Central

Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

2011-01-01

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

PubMed Central

Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

2016-01-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID:27382138

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses.

PubMed

Manzano, David; Andrade, Paola; Caudepón, Daniel; Altabella, Teresa; Arró, Montserrat; Ferrer, Albert

2016-09-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. © 2016 American Society of Plant Biologists. All rights reserved.

cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea.

PubMed

Zhu, Changfu; Yamamura, Saburo; Koiwa, Hiroyuki; Nishihara, Masashiro; Sandmann, Gerhard

2002-02-01

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes.

PubMed

Sarker, Lukman S; Demissie, Zerihun A; Mahmoud, Soheil S

2013-11-01

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae.

PubMed

Liu, Jidong; Zhang, Weiping; Du, Guocheng; Chen, Jian; Zhou, Jingwen

2013-12-01

Monoterpene geraniol, a compound obtained from aromatic plants, has wide applications. In this study, geraniol was synthesized in Saccharomyces cerevisiae through the introduction of geraniol synthase. To increase geraniol production, the mevalonate pathway in S. cerevisiae was genetically manipulated to enhance the supply of geranyl diphosphate, a substrate used for the biosynthesis of geraniol. Identification and optimization of the key regulatory points in the mevalonate pathway in S. cerevisiae increased geraniol production to 36.04 mg L(-1). The results obtained revealed that the IDI1-encoded isopentenyl diphosphate isomerase is a rate-limiting enzyme in the biosynthesis of geraniol in S. cerevisiae, and overexpression of MAF1, a negative regulator in tRNA biosynthesis, is another effective method to increase geraniol production in S. cerevisiae. Copyright © 2013 Elsevier B.V. All rights reserved.

Coexpression Analysis Identifies Two Oxidoreductases Involved in the Biosynthesis of the Monoterpene Acid Moiety of Natural Pyrethrin Insecticides in Tanacetum cinerariifolium1[OPEN

PubMed Central

Moghe, Gaurav D.

2018-01-01

Flowers of Tanacetum cinerariifolium produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. TcCDS produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencing-based approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that TcADH2 encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while TcALDH1 encodes an enzyme that oxidizes trans-chrysanthemal into trans-chrysanthemic acid. Transient coexpression of TcADH2 and TcALDH1 together with TcCDS in Nicotiana benthamiana leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host. PMID:29122986

Statin-induced muscle damage and atrogin-1 induction is the result of a geranylgeranylation defect

PubMed Central

Cao, Peirang; Hanai, Jun-ichi; Tanksale, Preeti; Imamura, Shintaro; Sukhatme, Vikas P.; Lecker, Stewart H.

2009-01-01

Statins are widely used to treat hypercholesterolemia but can lead to a number of side effects in muscle, including rhabdomyolysis. Our recent findings implicated the induction of atrogin-1, a gene required for the development of muscle atrophy, in statin-induced muscle damage. Since statins inhibit many biochemical reactions besides cholesterol synthesis, we sought to define the statin-inhibited pathways responsible for atrogin-1 expression and muscle damage. We report here that lovastatin-induced atrogin-1 expression and muscle damage in cultured mouse myotubes and zebrafish can be prevented in the presence of geranylgeranol but not farnesol. Further, inhibitors of the transfer of geranylgeranyl isoprene units to protein targets cause statin muscle damage and atrogin-1 induction in cultured cells and in fish. These findings support the concept that dysfunction of small GTP-binding proteins lead to statin-induced muscle damage since these molecules require modification by geranylgeranyl moieties for their cellular localization and activity. Collectively, our animal and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be regulated by novel signaling pathways.—Cao, P., Hanai, J., Tanksale, P., Imamura, S., Sukhatme, V. P., Lecker, S. H. Statin-induced muscle damage and atrogin-1 induction is the result of a geranylgeranylation defect. PMID:19406843

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression

PubMed Central

Moreno, J.C.; Cerda, A.; Simpson, K.; Lopez-Diaz, I.; Carrera, E; Handford, M.; Stange, C.

2016-01-01

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. PMID:26893492

The first structure of a bacterial diterpene cyclase: CotB2.

PubMed

Janke, Ronja; Görner, Christian; Hirte, Max; Brück, Thomas; Loll, Bernhard

2014-06-01

Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

Differential transcriptional control of the two tRNA(fMet) genes of Escherichia coli K-12.

PubMed

Nagase, T; Ishii, S; Imamoto, F

1988-07-15

The metZ gene of Escherichia coli, which encodes the tRNA(f1Met), was cloned. Using the nucleotide sequence, in vitro transcription, and S1 nuclease mapping analyses, we identified the promoter region, transcriptional start point, the two tandem tRNA(f1Met) structural genes separated by an intergenic space of 33 bp, and the two Rho-independent transcriptional termination sites, in that order. We compared the promoter region of the metZ gene with that of the metY gene, which encodes the tRNA(f2Met) and is located in the promoter-proximal portion of the nusA operon. A G + C-rich sequence (5'-GCGCATCCAC-3'), similar to the corresponding sequence of the rrn promoters that are under stringent control, was found between the Pribnow box and the transcriptional start point of the metZ promoter, but not in the metY promoter region. We therefore examined the effect of guanosine 3'-diphosphate, 5'-diphosphate (ppGpp), the chemical mediator of stringent control, and found that ppGpp inhibited the transcription of the metZ gene, but not that of the metY gene. These data suggested that the promoters for metZ and metY have different physiological functions and are regulated by different mechanisms.

Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis

PubMed Central

Gutkowska, Malgorzata; Wnuk, Marta; Nowakowska, Julita; Lichocka, Malgorzata; Stronkowski, Michal M.; Swiezewska, Ewa

2015-01-01

Rab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties. Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1–43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system. PMID:25316062

Characterization of Geraniol Synthase from the Peltate Glands of Sweet Basil1

PubMed Central

Iijima, Yoko; Gang, David R.; Fridman, Eyal; Lewinsohn, Efraim; Pichersky, Eran

2004-01-01

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 μm were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral. PMID:14657409

Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid.

PubMed Central

Kinsella, B T; Erdman, R A; Maltese, W A

1991-01-01

ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by [3H]geranylgeranyl instead of [3H]farnesyl in an in vitro assay. Gel-permeation chromatography of [3H]mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21(Leu-189) was also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21(Leu-189) with [3H]palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases. Images PMID:1924354

Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinsella, B.T.; Erdman, R.A.; Maltese, W.A.

ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. The authors changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by ({sup 3}H)geranylgeranyl instead of ({sup 3}H)farnesyl in an in vitro assay. Gel-permeation chromatography of ({sup 3}H)mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21 (Leu-189) wasmore » also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21 (Leu-189) with ({sup 3}H) palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases.« less

Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols.

PubMed

Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2009-04-01

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression.

PubMed

Moreno, J C; Cerda, A; Simpson, K; Lopez-Diaz, I; Carrera, E; Handford, M; Stange, C

2016-04-01

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Identifying Potential Therapeutics for Osteoporosis by Exploiting the Relationship between Mevalonate Pathway and Bone Metabolism.

PubMed

Wan Hasan, Wan Nuraini; Chin, Kok-Yong; Jolly, James Jam; Abd Ghafar, Norzana; Soelaiman, Ima Nirwana

2018-04-23

Osteoporosis is a silent skeletal disease characterized by low bone mass and destruction of skeletal microarchitecture, leading to an increased fracture risk. This occurs due to an imbalance in bone remodelling, whereby the rate of bone resorption is greater than bone formation. Mevalonate pathway, previously known to involve in cholesterol synthesis, is an important regulatory pathway for bone remodelling. This review aimed to provide an overview of the relationship between mevalonate pathway and bone metabolism, as well as agents which act through this pathway to achieve their therapeutic potential. Mevalonate pathway produces farnesyl pyrophosphate and geranylgeranyl pyrophosphate essential in protein prenylation. An increase in protein prenylation favours bone resorption over bone formation. Non-nitrogen containing bisphosphonates inhibit farnesyl diphosphate synthase which produces farnesyl pyrophosphate. They are used as the first line therapy for osteoporosis. Statins, a well-known class of cholesterol-lowering agents, inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-determining enzyme in the mevalonate pathway. It was shown to increase bone mineral density and prevent fracture in humans. Tocotrienol is a group of vitamin E commonly found in palm oil, rice bran and annatto bean. It causes degradation of HMG-CoA reductase. Many studies demonstrated that tocotrienol prevented bone loss in animal studies but its efficacy has not been tested in humans. mevalonate pathway can be exploited to develop effective antiosteoporosis agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

PubMed

Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2010-07-01

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis

PubMed Central

Tanaka, Ryouichi; Rothbart, Maxi; Oka, Seiko; Takabayashi, Atsushi; Takahashi, Kaori; Shibata, Masaru; Myouga, Fumiyoshi; Motohashi, Reiko; Shinozaki, Kazuo; Grimm, Bernhard

2010-01-01

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase. PMID:20823244

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking

PubMed Central

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. PMID:22960045

Active-site-directed irreversible inhibitors of isopentenyl diphosphate isomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muhlbacher, M.

1987-01-01

Seven analogues of isopentenyl diphosphate, containing fluorine, epoxy, or ammonium functionalities were found to irreversibly inhibit isopentenyl diphosphate:dimethylallyl diphosphate isomerase isolated from the mold Claviceps purpurea. The mechanism of their inhibition of isomerase was studied. Syntheses of 3-(fluoromethyl)-3-buten-1-yl diphosphate, 2-dimethylamino-1-ethyl diphosphate, 3,4-epoxy-3-methyl-1-butyl diphosphate, 3,4,-epoxy-1-butyl diphosphate, and 2,3-epoxy-3-methyl-1-butyl diphosphate were developed and carried out in high overall yield affording 100 mg quantities of the triammonium diphosphate salts. Radiolabeled materials of these analogues with {sup 3}H, {sup 14}C, and {sup 32}P at appropriate positions were also prepared. Inactivation kinetics, substrate protection studies, and labeling experiments demonstrated that the analogues interact stoichiometrically withmore » the active-site of isomerase. Radioactive enzyme-inactivator complexes were isolated, that are stable to extended dialysis and chaotropic reagents. The complexes resulting from inactivation of the enzyme by 3-(fluoromethyl)-3-buten-1-yl diphosphate and 3,4-epoxy-3-methyl-1-butyl diphosphate are stable to ion exchange chromatography and gel electrophoresis. Stoichiometric fluoride ion release occurs during inactivation of isomerase with 3-(fluoromethyl)-3-buten-1-yl diphosphate. The complexes are not stable to high concentrations of mixtures of 2-mercaptoethanol-sodium dodecyl sulfate. The radiolabeled 2-dimethylamino-1-ethyl diphosphate isomerase complex loses radioactivity almost instantaneously when treated with base. Partial fragmentation of the inactivator molecule was observed.« less

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

PubMed Central

Smith, Maria W.; Yamaguchi, Shinjiro; Ait-Ali, Tahar; Kamiya, Yuji

1998-01-01

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression. PMID:9847116

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10).

PubMed

Cluis, Corinne P; Ekins, Andrew; Narcross, Lauren; Jiang, Heng; Gold, Nicholas D; Burja, Adam M; Martin, Vincent J J

2011-11-01

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.

Intersubunit structure within heterodimers of medium-chain prenyl diphosphate synthases. Formation of a hybrid-type heptaprenyl diphosphate synthase.

PubMed

Koike-Takeshita, A; Koyama, T; Ogura, K

1998-10-01

Among prenyltransferases that catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphate to produce prenyl diphosphates with various chain lengths and stereochemistries, medium-chain prenyl diphosphate synthases are exceptional in that they comprise two dissociable heteromeric protein components. These components exist without binding with each other under physiological conditions, and neither of them has any prenyltransferase activity by itself. In order to elucidate the precise molecular mechanism underlying expression of the catalytic function by such a unique two-component system, we examined the possibility of forming a hybrid between two of the components of three different medium-chain prenyl diphosphate synthases, components I and II of heptaprenyl diphosphate synthase from Bacillus subtilis, components I' and II' of heptaprenyl diphosphate synthase from Bacillus stearothermophilus, and components A and B of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26. As a result, only the hybrid-type combination of component I and component II' gave distinct prenyltransferase activity. The hybrid-type enzyme catalyzed the synthesis of heptaprenyl diphosphate and showed moderate heat stability, which lay between those of the natural enzymes from B. subtilis and B. stearothermophilus. There is no possibility of forming a hybrid between the heptaprenyl and hexaprenyl diphosphate synthases.

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

PubMed

Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

2017-10-01

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

PubMed Central

Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

2001-01-01

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

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

PubMed Central

Bach, Thomas J

2013-01-01

We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

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

PubMed Central

Hartmann, Michael; Gas-Pascual, Elisabet; Hemmerlin, Andrea; Rohmer, Michel; Bach, Thomas J.

2015-01-01

In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system. PMID:26309725

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.

PubMed

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.

A cis-prenyltransferase from Methanosarcina acetivorans catalyzes both head-to-tail and nonhead-to-tail prenyl condensation.

PubMed

Ogawa, Takuya; Emi, Koh-Ichi; Koga, Kazushi; Yoshimura, Tohru; Hemmi, Hisashi

2016-06-01

Cis-prenyltransferase usually consecutively catalyzes the head-to-tail condensation reactions of isopentenyl diphosphate to allylic prenyl diphosphate in the production of (E,Z-mixed) polyprenyl diphosphate, which is the precursor of glycosyl carrier lipids. Some recently discovered homologs of the enzyme, however, catalyze the nonhead-to-tail condensation reactions between allylic prenyl diphosphates. In this study, we characterize a cis-prenyltransferase homolog from a methanogenic archaeon, Methanosarcina acetivorans, to obtain information on the biosynthesis of the glycosyl carrier lipids within it. This enzyme catalyzes both head-to-tail and nonhead-to-tail condensation reactions. The kinetic analysis shows that the main reaction of the enzyme is consecutive head-to-tail prenyl condensation reactions yielding polyprenyl diphosphates, while the chain lengths of the major products seem shorter than expected for the precursor of glycosyl carrier lipids. On the other hand, a subsidiary reaction of the enzyme, i.e., nonhead-to-tail condensation between dimethylallyl diphosphate and farnesyl diphosphate, gives a novel diterpenoid compound, geranyllavandulyl diphosphate. © 2016 Federation of European Biochemical Societies.

Synthesis and Evaluation of Chlorinated Substrate Analogues for Farnesyl Diphosphate Synthase

PubMed Central

Heaps, Nicole A.; Poulter, C. Dale

2011-01-01

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a co-substrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of KM3-ClIPP (with DMAPP) and KM3-ClDMAPP (with IPP) were similar to those for IPP and DMAPP, however values of kcat for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate. PMID:21344952

Carotenoid Biosynthetic Pathways Are Regulated by a Network of Multiple Cascades of Alternative Sigma Factors in Azospirillum brasilense Sp7.

PubMed

Rai, Ashutosh Kumar; Dubey, Ashutosh Prakash; Kumar, Santosh; Dutta, Debashis; Mishra, Mukti Nath; Singh, Bhupendra Narain; Tripathi, Anil Kumar

2016-11-01

Carotenoids constitute an important component of the defense system against photooxidative stress in bacteria. In Azospirillum brasilense Sp7, a nonphotosynthetic rhizobacterium, carotenoid synthesis is controlled by a pair of extracytoplasmic function sigma factors (RpoEs) and their cognate zinc-binding anti-sigma factors (ChrRs). Its genome harbors two copies of the gene encoding geranylgeranyl pyrophosphate synthase (CrtE), the first critical step in the carotenoid biosynthetic pathway in bacteria. Inactivation of each of two crtE paralogs found in A. brasilense caused reduction in carotenoid content, suggesting their involvement in carotenoid synthesis. However, the effect of crtE1 deletion was more pronounced than that of crtE2 deletion. Out of the five paralogs of rpoH in A. brasilense, overexpression of rpoH1 and rpoH2 enhanced carotenoid synthesis. Promoters of crtE2 and rpoH2 were found to be dependent on RpoH2 and RpoE1, respectively. Using a two-plasmid system in Escherichia coli, we have shown that the crtE2 gene of A. brasilense Sp7 is regulated by two cascades of sigma factors: one consisting of RpoE1and RpoH2 and the other consisting of RpoE2 and RpoH1. In addition, expression of crtE1 was upregulated indirectly by RpoE1 and RpoE2. This study shows, for the first time in any carotenoid-producing bacterium, that the regulation of carotenoid biosynthetic pathway involves a network of multiple cascades of alternative sigma factors. Carotenoids play a very important role in coping with photooxidative stress in prokaryotes and eukaryotes. Although extracytoplasmic function (ECF) sigma factors are known to directly regulate the expression of carotenoid biosynthetic genes in bacteria, regulation of carotenoid biosynthesis by one or multiple cascades of sigma factors had not been reported. This study provides the first evidence of the involvement of multiple cascades of sigma factors in the regulation of carotenoid synthesis in any bacterium by showing the regulation of a gene encoding geranylgeranyl pyrophosphate synthase (crtE2) by RpoE1→RpoH2→CrtE2 and RpoE2→RpoH1→CrtE2 cascades in A. brasilense It also provides an insight into existence of an additional cascade or cascades regulating expression of another paralog of crtE. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Carotenoid Biosynthetic Pathways Are Regulated by a Network of Multiple Cascades of Alternative Sigma Factors in Azospirillum brasilense Sp7

PubMed Central

Rai, Ashutosh Kumar; Dubey, Ashutosh Prakash; Kumar, Santosh; Dutta, Debashis; Mishra, Mukti Nath; Singh, Bhupendra Narain

2016-01-01

ABSTRACT Carotenoids constitute an important component of the defense system against photooxidative stress in bacteria. In Azospirillum brasilense Sp7, a nonphotosynthetic rhizobacterium, carotenoid synthesis is controlled by a pair of extracytoplasmic function sigma factors (RpoEs) and their cognate zinc-binding anti-sigma factors (ChrRs). Its genome harbors two copies of the gene encoding geranylgeranyl pyrophosphate synthase (CrtE), the first critical step in the carotenoid biosynthetic pathway in bacteria. Inactivation of each of two crtE paralogs found in A. brasilense caused reduction in carotenoid content, suggesting their involvement in carotenoid synthesis. However, the effect of crtE1 deletion was more pronounced than that of crtE2 deletion. Out of the five paralogs of rpoH in A. brasilense, overexpression of rpoH1 and rpoH2 enhanced carotenoid synthesis. Promoters of crtE2 and rpoH2 were found to be dependent on RpoH2 and RpoE1, respectively. Using a two-plasmid system in Escherichia coli, we have shown that the crtE2 gene of A. brasilense Sp7 is regulated by two cascades of sigma factors: one consisting of RpoE1and RpoH2 and the other consisting of RpoE2 and RpoH1. In addition, expression of crtE1 was upregulated indirectly by RpoE1 and RpoE2. This study shows, for the first time in any carotenoid-producing bacterium, that the regulation of carotenoid biosynthetic pathway involves a network of multiple cascades of alternative sigma factors. IMPORTANCE Carotenoids play a very important role in coping with photooxidative stress in prokaryotes and eukaryotes. Although extracytoplasmic function (ECF) sigma factors are known to directly regulate the expression of carotenoid biosynthetic genes in bacteria, regulation of carotenoid biosynthesis by one or multiple cascades of sigma factors had not been reported. This study provides the first evidence of the involvement of multiple cascades of sigma factors in the regulation of carotenoid synthesis in any bacterium by showing the regulation of a gene encoding geranylgeranyl pyrophosphate synthase (crtE2) by RpoE1→RpoH2→CrtE2 and RpoE2→RpoH1→CrtE2 cascades in A. brasilense. It also provides an insight into existence of an additional cascade or cascades regulating expression of another paralog of crtE. PMID:27551017

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate.

PubMed

Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

2015-01-01

Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

PubMed Central

Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

2015-01-01

Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 . PMID:26306151

Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis.

PubMed

Li, Chunhong; Ng, Ailing; Xie, Lifen; Mao, Huizhu; Qiu, Chengxiang; Srinivasan, Ramachandran; Yin, Zhongchao; Hong, Yan

2016-01-01

Casbene is a precursor to phorbol esters and down-regulating casbene synthase effectively reduces phorbol ester biosynthesis. Seed-specific reduction of phorbol ester (PE) helps develop Jatropha seed cake for animal nutrition. Phorbol esters (PEs) are diterpenoids present in some Euphorbiaceae family members like Jatropha curcas L. (Jatropha), a tropical shrub yielding high-quality oil suitable as feedstock for biodiesel and bio jet fuel. Jatropha seed contains up to 40 % of oil and can produce oil together with cake containing high-quality proteins. However, skin-irritating and cancer-promoting PEs make Jatropha cake meal unsuitable for animal nutrition and also raise some safety and environmental concerns on its planting and processing. Two casbene synthase gene (JcCASA163 and JcCASD168) homologues were cloned from Jatropha genome and both genes were highly expressed during seed development. In vitro functional analysis proved casbene synthase activity of JcCASA163 in converting geranylgeranyl diphosphate into casbene which has been speculated to be the precursor to PEs. A seed-specific promoter driving inverted repeats for RNAi interference targeting at either JcCASA163 or both genes could effectively down-regulate casbene synthase gene expression with concurrent marked reduction of PE level (by as much as 85 %) in seeds with no pleiotropic effects observed. Such engineered low PE in seed was heritable and co-segregated with the transgene. Our work implicated casbene synthase in Jatropha PE biosynthesis and provided evidence for casbene being the precursor for PEs. The success in reducing seed PE content through down-regulation of casbene synthase demonstrates the feasibility of intercepting PE biosynthesis in Jatropha seed to help address safety concerns on Jatropha plantation and seed processing and facilitate use of its seed protein for animal nutrition.

Endophytic Paecilomyces formosus LHL10 Augments Glycine max L. Adaptation to Ni-Contamination through Affecting Endogenous Phytohormones and Oxidative Stress

PubMed Central

Bilal, Saqib; Khan, Abdul L.; Shahzad, Raheem; Asaf, Sajjad; Kang, Sang-Mo; Lee, In-Jung

2017-01-01

This study investigated the Ni-removal efficiency of phytohormone-producing endophytic fungi Penicillium janthinellum, Paecilomyces formosus, Exophiala sp., and Preussia sp. Among four different endophytes, P. formosus LHL10 was able to tolerate up to 1 mM Ni in contaminated media as compared to copper and cadmium. P. formosus LHL10 was further assessed for its potential to enhance the phytoremediation of Glycine max (soybean) in response to dose-dependent increases in soil Ni (0.5, 1.0, and 5.0 mM). Inoculation with P. formosus LHL10 significantly increased plant biomass and growth attributes as compared to non-inoculated control plants with or without Ni contamination. LHL10 enhanced the translocation of Ni from the root to the shoot as compared to the control. In addition, P. formosus LHL10 modulated the physio-chemical apparatus of soybean plants during Ni-contamination by reducing lipid peroxidation and the accumulation of linolenic acid, glutathione, peroxidase, polyphenol oxidase, catalase, and superoxide dismutase. Stress-responsive phytohormones such as abscisic acid and jasmonic acid were significantly down-regulated in fungal-inoculated soybean plants under Ni stress. LHL10 Ni-remediation potential can be attributed to its phytohormonal synthesis related genetic makeup. RT-PCR analysis showed the expression of indole-3-acetamide hydrolase, aldehyde dehydrogenase for indole-acetic acid and geranylgeranyl-diphosphate synthase, ent-kaurene oxidase (P450-4), C13-oxidase (P450-3) for gibberellins synthesis. In conclusion, the inoculation of P. formosus can significantly improve plant growth in Ni-polluted soils, and assist in improving the phytoremediation abilities of economically important crops. PMID:28611799

Jasmonic Acid Is a Key Regulator of Spider Mite-Induced Volatile Terpenoid and Methyl Salicylate Emission in Tomato1[w

PubMed Central

Ament, Kai; Kant, Merijn R.; Sabelis, Maurice W.; Haring, Michel A.; Schuurink, Robert C.

2004-01-01

The tomato (Lycopersicon esculentum) mutant def-1, which is deficient in induced jasmonic acid (JA) accumulation upon wounding or herbivory, was used to study the role of JA in the direct and indirect defense responses to phytophagous mites (Tetranychus urticae). In contrast to earlier reports, spider mites laid as many eggs and caused as much damage on def-1 as on wild-type plants, even though def-1 lacked induction of proteinase inhibitor activity. However, the hatching-rate of eggs on def-1 was significantly higher, suggesting that JA-dependent direct defenses enhanced egg mortality or increased the time needed for embryonic development. As to gene expression, def-1 had lower levels of JA-related transcripts but higher levels of salicylic acid (SA) related transcripts after 1 d of spider mite infestation. Furthermore, the indirect defense response was absent in def-1, since the five typical spider mite-induced tomato-volatiles (methyl salicylate [MeSA], 4,8,12-trimethyltrideca-1,3,7,11-tetraene [TMTT], linalool, trans-nerolidol, and trans-β-ocimene) were not induced and the predatory mite Phytoseiulus persimilis did not discriminate between infested and uninfested def-1 tomatoes as it did with wild-type tomatoes. Similarly, the expression of the MeSA biosynthetic gene salicylic acid methyltransferase (SAMT) was induced by spider mites in wild type but not in def-1. Exogenous application of JA to def-1 induced the accumulation of SAMT and putative geranylgeranyl diphosphate synthase transcripts and restored MeSA- and TMTT-emission upon herbivory. JA is therefore necessary to induce the enzymatic conversion of SA into MeSA. We conclude that JA is essential for establishing the spider mite-induced indirect defense response in tomato. PMID:15310835

Endophytic Paecilomyces formosus LHL10 Augments Glycine max L. Adaptation to Ni-Contamination through Affecting Endogenous Phytohormones and Oxidative Stress.

PubMed

Bilal, Saqib; Khan, Abdul L; Shahzad, Raheem; Asaf, Sajjad; Kang, Sang-Mo; Lee, In-Jung

2017-01-01

This study investigated the Ni-removal efficiency of phytohormone-producing endophytic fungi Penicillium janthinellum , Paecilomyces formosus , Exophiala sp., and Preussia sp. Among four different endophytes, P. formosus LHL10 was able to tolerate up to 1 mM Ni in contaminated media as compared to copper and cadmium. P. formosus LHL10 was further assessed for its potential to enhance the phytoremediation of Glycine max (soybean) in response to dose-dependent increases in soil Ni (0.5, 1.0, and 5.0 mM). Inoculation with P. formosus LHL10 significantly increased plant biomass and growth attributes as compared to non-inoculated control plants with or without Ni contamination. LHL10 enhanced the translocation of Ni from the root to the shoot as compared to the control. In addition, P. formosus LHL10 modulated the physio-chemical apparatus of soybean plants during Ni-contamination by reducing lipid peroxidation and the accumulation of linolenic acid, glutathione, peroxidase, polyphenol oxidase, catalase, and superoxide dismutase. Stress-responsive phytohormones such as abscisic acid and jasmonic acid were significantly down-regulated in fungal-inoculated soybean plants under Ni stress. LHL10 Ni-remediation potential can be attributed to its phytohormonal synthesis related genetic makeup. RT-PCR analysis showed the expression of indole-3-acetamide hydrolase , aldehyde dehydrogenase for indole-acetic acid and geranylgeranyl-diphosphate synthase , ent-kaurene oxidase ( P450-4 ), C13-oxidase ( P450-3 ) for gibberellins synthesis. In conclusion, the inoculation of P. formosus can significantly improve plant growth in Ni-polluted soils, and assist in improving the phytoremediation abilities of economically important crops.

Prenyl alcohol production by expression of exogenous isopentenyl diphosphate isomerase and farnesyl diphosphate synthase genes in Escherichia coli.

PubMed

Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2009-01-01

Isopentenyl diphosphate isomerase (idi) and farnesyl diphosphate synthase (ispA) genes were overexpressed in Escherichia coli. The resulting transformant showed 6.8-fold higher production of farnesol (389 microg/l). In a similar manner, overexpression of idi and mutated ispA led to high production of geranylgeraniol (128 microg/l).

Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gabelli,S.; McLellan, J.; Montalvetti, A.

2006-01-01

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformationalmore » change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.« less

Characterization of a Root-Specific Arabidopsis Terpene Synthase Responsible for the Formation of the Volatile Monoterpene 1,8-Cineole1

PubMed Central

Chen, Feng; Ro, Dae-Kyun; Petri, Jana; Gershenzon, Jonathan; Bohlmann, Jörg; Pichersky, Eran; Tholl, Dorothea

2004-01-01

Arabidopsis is emerging as a model system to study the biochemistry, biological functions, and evolution of plant terpene secondary metabolism. It was previously shown that the Arabidopsis genome contains over 30 genes potentially encoding terpene synthases (TPSs). Here we report the characterization of a monoterpene synthase encoded by two identical, closely linked genes, At3g25820 and At3g25830. Transcripts of these genes were detected almost exclusively in roots. An At3g25820/At3g25830 cDNA was expressed in Escherichia coli, and the protein thus produced was shown to catalyze the formation of 10 volatile monoterpenes from geranyl diphosphate, with 1,8-cineole predominating. This protein was therefore designated AtTPS-Cin. The purified recombinant AtTPS-Cin displayed similar biochemical properties to other known monoterpene synthases, except for a relatively low Km value for geranyl diphosphate of 0.2 μm. At3g25820/At3g25830 promoter activity, measured with a β-glucuronidase (GUS) reporter gene, was primarily found in the epidermis, cortex, and stele of mature primary and lateral roots, but not in the root meristem or the elongation zone. Although the products of AtTPS-Cin were not detected by direct extraction of plant tissue, the recent report of 1,8-cineole as an Arabidopsis root volatile (Steeghs M, Bais HP, de Gouw J, Goldan P, Kuster W, Northway M, Fall R, Vivanco JM [2004] Plant Physiol 135: 47–58) suggests that the enzyme products may be released into the rhizosphere rather than accumulated. Among Arabidopsis TPSs, AtTPS-Cin is most similar to the TPS encoded by At3g25810, a closely linked gene previously shown to be exclusively expressed in flowers. At3g25810 TPS catalyzes the formation of a set of monoterpenes that is very similar to those produced by AtTPS-Cin, but its major products are myrcene and (E)-β-ocimene, and it does not form 1,8-cineole. These data demonstrate that divergence of organ expression pattern and product specificity are ongoing processes within the Arabidopsis TPS family. PMID:15299125

HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik, E-mail: henrik.thorlacius@med.lu.se

2014-03-28

Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluatemore » CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via inhibition of geranylgeranylation and RhoA activation. Thus, statins, such as simvastatin, might be effective tools to antagonize CCL17-dependent migration and metastasis of colon cancer cells.« less

Statins inhibit blastocyst formation by preventing geranylgeranylation

PubMed Central

Alarcon, Vernadeth B.; Marikawa, Yusuke

2016-01-01

STUDY HYPOTHESIS Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway and prescription drugs that treat hypercholesterolemia, compromise preimplantation mouse development via modulation of HIPPO signaling. STUDY FINDING HMG-CoA reductase activity is required for trophectoderm specification, namely blastocyst cavity formation and Yes-associated protein (YAP) nuclear localization, through the production of isoprenoid geranylgeranyl pyrophosphate (GGPP) and the action of geranylgeranyl transferase. WHAT IS KNOWN ALREADY Previous studies have shown that treatment of mouse embryos with mevastatin prevents blastocyst formation, but how HMG-CoA reductase is involved in preimplantation development is unknown. HIPPO signaling regulates specification of the trophectoderm lineage of the mouse blastocyst by controlling the nuclear localization of YAP. In human cell lines, the mevalonate pathway regulates YAP to mediate self-renewal and survival through geranylgeranylation of RHO proteins. These studies suggest that in preimplantation development, statins may act through HIPPO pathway to interfere with trophectoderm specification and thereby inhibit blastocyst formation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Eight-cell stage (E2.5) mouse embryos were treated in hanging drop culture with chemical agents, namely statins (lovastatin, atorvastatin, cerivastatin and pravastatin), mevalonic acid (MVA), cholesterol, squalene, farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranylgeranyltransferase inhibitor GGTI-298, RHO inhibitor I, and squalene synthase inhibitor YM-53601, up to the late blastocyst stage (E4.5). Efficiency of blastocyst formation was assessed based on gross morphology and the measurement of the cavity size using an image analysis software. Effects on cell lineages and HIPPO signaling were analyzed using immunohistochemistry with confocal microscopy based on the expression patterns of the lineage-specific markers and the nuclear accumulation of YAP. Effects on cell lineages were also examined by quantitative RT–PCR based on the transcript levels of the lineage-specific marker genes. Data were analyzed using one-way ANOVA and two-sample t-test. MAIN RESULTS AND THE ROLE OF CHANCE All four statins examined inhibited blastocyst formation. The adverse impact of statins was rescued by supplementation of MVA (P < 0.01) or GGPP (P < 0.01) but not squalene nor cholesterol. Blastocyst formation was also prevented by GGTI-298 (P < 0.01). These results indicate that HMG-CoA reductase activity is required for blastocyst formation mainly through the production of GGPP but not cholesterol. Inhibition of RHO proteins, known targets of geranylgeranylation, impaired blastocyst formation, which was not reversed by GGPP supplementation. Nuclear localization of YAP was diminished by statin treatment but fully restored by supplementation of MVA (P < 0.01) or GGPP (P < 0.01). This suggests that HIPPO signaling is regulated by GGPP-dependent mechanisms, possibly geranylgeranylation of RHO, to enable trophectoderm formation. YM-53601 prevented blastocyst formation (P < 0.01), but its adverse impact was not rescued by supplementation of squalene or cholesterol, suggesting that squalene synthesis inhibition was not the cause of blastocyst defects. LIMITATIONS, REASONS FOR CAUTION Analyses were conducted on embryos cultured ex vivo, but they enable the determination of specific concentrations that impair embryo development which can be compared with drug concentrations in the reproductive tract when testing in vivo impact of statins through animal experimentations. Also, analyses were conducted in only one species, the mouse. Epidemiological studies on the effects of various types of statins on the fertility of women are necessary. WIDER IMPLICATIONS OF THE FINDINGS Our study reveals how the mevalonate pathway is required for blastocyst formation and intersects with HIPPO pathway to provide a mechanistic basis for the embryotoxic effect of statins. This bears relevance for women who are taking statins while trying to conceive, since statins have potential to prevent the conceptus from reaching the blastocyst stage and to cause early conceptus demise. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by grants from the George F. Straub Trust of the Hawaii Community Foundation (13ADVC-60315 to V.B.A.) and the National Institutes of Health, USA (P20GM103457 to V.B.A.). The authors have no conflict of interest to declare. PMID:26908642

Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

PubMed

Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

2016-11-01

2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104.

PubMed

Torgov, Vladimir; Danilov, Leonid; Utkina, Natalia; Veselovsky, Vladimir; Brockhausen, Inka

2017-12-01

Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P 1 -(11-phenoxyundecyl)-P 2 - (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P 1 -(11-phenoxyundecyl)-P 2 -(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as a secondary metabolite from D-fructose-1,6-diphosphate metabolism by Zygosaccharomyces rouxii.

PubMed

Dahlen, T; Hauck, T; Wein, M; Schwab, W

2001-01-01

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is an important aroma compound found in many fruits such as strawberries and pineapples and it is also produced by the soy-sauce-fermenting yeast Zygosaccharomyces rouxii after the addition of d-fructose-1,6-diphosphate to yeast-peptone-dextrose nutrient media. Dilute DMHF solutions exhibit a strawberry-like flavor while DMHF concentrates have a caramel-like aroma. In media containing D-fructose-1,6-diphosphate as the sole carbon source, growth of Z. rouxii and formation of DMHF were not observed. Although Z. rouxii cells grew in media with D-glucose as the sole carbon source, DMHF was only produced when media were supplemented with D-fructose-1,6-diphosphate. The DMHF concentration always correlated with the yeast cell count and D-fructose-1,6-diphosphate concentration. Addition of CaCl2 (up to 50 g.l(-1)) led to a higher DMHF concentration. Addition of Na2SO3 reduced the growth of Z. rouxii and inhibited DMHF formation. The amount of DMHF formed by Z. rouxii was not significantly affected by the addition of KH2PO4. DMHF concentrations of 5 and 10 g.l(-1) partially and completely inhibited the growth of Z. rouxii cells, respectively. Only the singly labeled furanone was formed after the addition of 1-13C-D-fructose-1,6-diphosphate to the medium. However, unlabeled DMHF was formed in the presence of (13)C(6)-D-glucose. Therefore, the carbons of the furanone originate exclusively from exogenously supplied D-fructose-1,6-diphosphate as no exchange with the internal pool of D-fructose-1,6-diphosphate occurs. This implies that DMHF is a secondary metabolite of Z. rouxii formed from D-fructose-1,6-diphosphate. We assume that at least the first step of the metabolism of D-fructose-1,6-diphosphate takes place in the cell wall or membrane of the yeast.

An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

PubMed Central

Dozier, Jonathan K; Distefano, Mark D

2012-01-01

Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays use either radiolabeled substrates and are discontinuous, or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format and that it can reproduce IC50 values for several previously reported FDPS inhibitors. This new method offers a simple, safe and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target. PMID:22085443

Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

NASA Technical Reports Server (NTRS)

Mar, A.; Dworkin, J.; Oro, J.

1987-01-01

Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

Phosphorylation of mononucleotides and formation of cytidine 5'-diphosphate-choline and sugar nucleotides by respiration-deficient mutants of yeasts.

PubMed Central

Kimura, A; Hirose, K; Kariya, Y; Nagai, S

1976-01-01

Respiration-deficient mutants (Rho-, petite) of Saccharomyces carlsbergensis were obtained by treatment with trypaflavin (euflavine). Dried cells of these mutants phosphorylated mononucleotides to their triphosphates and further formed not only cytidine 5'-diphosphate-choline, but also sugar nucleotides, such as uridine 5'-diphosphate-glucose, guanosine 5'-diphosphate-mannose, etc. The activities were the same or slightly greater than those of the wild strain. These results showed that energy (adenosine 5'-triphosphate) necessary for phosphorylation of mononucleotides was sufficiently supplied by the glycolysis system. PMID:1245470

Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

PubMed

Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

2016-10-19

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED 50 ∼0.15 μg mL -1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fruit color mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

USDA-ARS?s Scientific Manuscript database

Isoprenoids are a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized in the cytoplasm from mevalonic acid via the “MVA pathway” a...

Properties of ribulose diphosphate carboxylase immobilized on porous glass

NASA Technical Reports Server (NTRS)

Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

1974-01-01

Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

PubMed Central

Puts, Johan; de Groot, Monique; Haex, Martin; Jakobs, Bernadette

2015-01-01

Background Vitamin B1 (thiamine-diphosphate) and B6 (pyridoxal-5’phosphate) are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due to malnutrition, in severe alcoholism and in gastrointestinal compromise due to bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC) based methods include the need for derivatization and long analysis time. We developed an Ultra High Performance Liquid Chromatography Tandem Mass spectrometry (UHPLC-MS/MS) assay with internal standards for simultaneous measurement of underivatized thiamine-diphosphate and pyridoxal-5’phosphate without use of ion pairing reagent. Methods Whole blood, deproteinized with perchloric acid, containing deuterium labelled internal standards thiamine-diphosphate(thiazole-methyl-D3) and pyridoxal-5’phosphate(methyl-D3), was analyzed by UHPLC-MS/MS. The method was validated for imprecision, linearity, recovery and limit of quantification. Alternate (quantitative) method comparisons of the new versus currently used routine HPLC methods were established with Deming regression. Results Thiamine-diphosphate and pyridoxal-5’phosphate were measured within 2.5 minutes instrumental run time. Limits of detection were 2.8 nmol/L and 7.8 nmol/L for thiamine-diphosphate and pyridoxal-5’phosphate respectively. Limit of quantification was 9.4 nmol/L for thiamine-diphosphate and 25.9 nmol/L for pyridoxal-5’phosphate. The total imprecision ranged from 3.5–7.7% for thiamine-diphosphate (44–157 nmol/L) and 6.0–10.4% for pyridoxal-5’phosphate (30–130 nmol/L). Extraction recoveries were 101–102% ± 2.5% (thiamine-diphosphate) and 98–100% ± 5% (pyridoxal-5’phosphate). Deming regression yielded slopes of 0.926 and 0.990 in patient samples (n = 282) and national proficiency testing samples (n = 12) respectively, intercepts of +3.5 and +3 for thiamine-diphosphate (n = 282 and n = 12) and slopes of 1.04 and 0.84, intercepts of -2.9 and +20 for pyridoxal-5’phosphate (n = 376 and n = 12). Conclusion The described UHPLC-MS/MS method allows simultaneous determination of underivatized thiamine-diphosphate and pyridoxal-5’phosphate in whole blood without intensive sample preparation. PMID:26134844

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: Construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

DOE Office of Scientific and Technical Information (OSTI.GOV)

Street, I.P.; Poulter, C.D.

1990-08-14

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinantmore » IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with (4-{sup 3}H)FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.« less

Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album

PubMed Central

Srivastava, Prabhakar Lal; Daramwar, Pankaj P.; Krithika, Ramakrishnan; Pandreka, Avinash; Shankar, S. Shiva; Thulasiram, Hirekodathakallu V.

2015-01-01

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, β-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems. PMID:25976282

Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album.

PubMed

Srivastava, Prabhakar Lal; Daramwar, Pankaj P; Krithika, Ramakrishnan; Pandreka, Avinash; Shankar, S Shiva; Thulasiram, Hirekodathakallu V

2015-05-15

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, β-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems.

Subcellular localization and compartmentation of thiamine derivatives in rat brain.

PubMed

Bettendorff, L; Wins, P; Lesourd, M

1994-05-26

The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate.

Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency.

PubMed

Tricarico, Paola Maura; Romeo, Alessandra; Gratton, Rossella; Crovella, Sergio; Celsi, Fulvio

2017-01-01

Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. MVK mutants' over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation. © 2017 The Author(s)Published by S. Karger AG, Basel.

Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

PubMed

Vickers, Claudia E; Possell, Malcolm; Nicholas Hewitt, C; Mullineaux, Philip M

2010-07-01

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

PubMed

Fujimura, Tsutomu; Esteban, Rosa

2016-10-01

The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm. © 2016 John Wiley & Sons Ltd.

Carotenoid Biosynthesis in Fusarium

PubMed Central

Avalos, Javier; Pardo-Medina, Javier; Parra-Rivero, Obdulia; Ruger-Herreros, Macarena; Rodríguez-Ortiz, Roberto; Hornero-Méndez, Dámaso; Limón, María Carmen

2017-01-01

Many fungi of the genus Fusarium stand out for the complexity of their secondary metabolism. Individual species may differ in their metabolic capacities, but they usually share the ability to synthesize carotenoids, a family of hydrophobic terpenoid pigments widely distributed in nature. Early studies on carotenoid biosynthesis in Fusarium aquaeductuum have been recently extended in Fusarium fujikuroi and Fusarium oxysporum, well-known biotechnological and phytopathogenic models, respectively. The major Fusarium carotenoid is neurosporaxanthin, a carboxylic xanthophyll synthesized from geranylgeranyl pyrophosphate through the activity of four enzymes, encoded by the genes carRA, carB, carT and carD. These fungi produce also minor amounts of β-carotene, which may be cleaved by the CarX oxygenase to produce retinal, the rhodopsin’s chromophore. The genes needed to produce retinal are organized in a gene cluster with a rhodopsin gene, while other carotenoid genes are not linked. In the investigated Fusarium species, the synthesis of carotenoids is induced by light through the transcriptional induction of the structural genes. In some species, deep-pigmented mutants with up-regulated expression of these genes are affected in the regulatory gene carS. The molecular mechanisms underlying the control by light and by the CarS protein are currently under investigation. PMID:29371556

Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

NASA Technical Reports Server (NTRS)

Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

1968-01-01

Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

D-tagatose 1,6-diphosphate aldolase from lactic streptococci: purification, properties, and use in measuring intracellular tagatose 1,6-diphosphate.

PubMed Central

Crow, V L; Thomas, T D

1982-01-01

Two D-ketohexose 1,6-diphosphate aldolases are present in Streptococcus cremoris E8 and S. lactis C10. One aldolase, which was induced by growth on either lactose or galactose, was active with both tagatose 1,6-diphosphate (TDP) and fructose 1,6-diphosphate (FDP), having a lower Km and a higher Vmax with TDP as the substrate. This enzyme, named TDP aldolase, had properties typical of a class I aldolase, being insensitive to EDTA and showing substrate-dependent inactivation by sodium borohydride. Sodium dodecyl sulfate-gel electrophoresis indicated a subunit molecular weight of 34,500. The amino acid composition of TDP aldolase is reported. When the enzyme was incubated with either triose phosphates or FDP, the equilibrium mixture contained an FDP/TDP ratio of 6.9:1. The other aldolase, which had properties typical of a class II aldolase, showed activity with FDP but not with TDP. The intracellular TDP concentration, measured with the purified TDP aldolase, was 0.4 to 4.0 mM in cells growing on lactose or galactose and was lower (0 to 1.0 mM) in cells growing on glucose. The intracellular concentration of FDP was always higher than that of TDP. The role of ketohexose diphosphates in the regulation of end product fermentation by lactic streptococci is discussed. PMID:6807956

Dependence of the product chain-length on detergents for long-chain E-polyprenyl diphosphate synthases

PubMed Central

Pan, Jian-Jung; Ramamoorthy, Gurusankar; Poulter, C. Dale

2013-01-01

Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and non-ionic detergents, we discovered considerable variability. Examination of a series of non-ionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain lengths. PMID:23802587

Molecular regulation of santalol biosynthesis in Santalum album L.

PubMed

Rani, Arti; Ravikumar, Puja; Reddy, Manjunatha Damodara; Kush, Anil

2013-09-25

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 - a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 - an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis. © 2013.

Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis*

PubMed Central

Yeo, Yun-Soo; Nybo, S. Eric; Chittiboyina, Amar G.; Weerasooriya, Aruna D.; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C. Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A. Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A.; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A.; Coates, Robert M.; Watt, David S.; Chappell, Joe

2013-01-01

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes. PMID:23243312

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

PubMed

Yeo, Yun-Soo; Nybo, S Eric; Chittiboyina, Amar G; Weerasooriya, Aruna D; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A; Coates, Robert M; Watt, David S; Chappell, Joe

2013-02-01

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Dark conditions enhance aluminum tolerance in several rice cultivars via multiple modulations of membrane sterols.

PubMed

Wagatsuma, Tadao; Maejima, Eriko; Watanabe, Toshihiro; Toyomasu, Tomonobu; Kuroda, Masaharu; Muranaka, Toshiya; Ohyama, Kiyoshi; Ishikawa, Akifumi; Usui, Masami; Hossain Khan, Shahadat; Maruyama, Hayato; Tawaraya, Keitaro; Kobayashi, Yuriko; Koyama, Hiroyuki

2018-01-23

Aluminum-sensitive rice (Oryza sativa L.) cultivars showed increased Al tolerance under dark conditions, because less Al accumulated in the root tips (1 cm) under dark than under light conditions. Under dark conditions, the root tip concentration of total sterols, which generally reduce plasma membrane permeabilization, was higher in the most Al-sensitive japonica cultivar, Koshihikari (Ko), than in the most Al-tolerant cultivar, Rikuu-132 (R132), but the phospholipid content did not differ between the two. The Al treatment increased the proportion of stigmasterol (which has no ability to reduce membrane permeabilization) out of total sterols similarly in both cultivars under light conditions, but it decreased more in Ko under dark conditions. The carotenoid content in the root tip of Al-treated Ko was significantly lower under dark than under light conditions, indicating that isopentenyl diphosphate transport from the cytosol to plastids was decreased under dark conditions. HMG2 and HMG3 (encoding the key sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase) transcript levels in the root tips were enhanced under dark conditions. We suggest that the following mechanisms contribute to the increase in Al tolerance under dark conditions: inhibition of stigmasterol formation to retain membrane integrity; greater partitioning of isopentenyl diphosphate for sterol biosynthesis; and enhanced expression of HMGs to increase sterol biosynthesis. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is notmore » involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.« less

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase*S⃞

PubMed Central

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-01-01

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes. PMID:19158086

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

PubMed

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-04-03

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

PubMed

Zhu, J K; Bressan, R A; Hasegawa, P M

1993-09-15

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo.

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

PubMed Central

Zhu, J K; Bressan, R A; Hasegawa, P M

1993-01-01

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:8378331

Gain and Loss of Fruit Flavor Compounds Produced by Wild and Cultivated Strawberry Species

PubMed Central

Aharoni, Asaph; Giri, Ashok P.; Verstappen, Francel W.A.; Bertea, Cinzia M.; Sevenier, Robert; Sun, Zhongkui; Jongsma, Maarten A.; Schwab, Wilfried; Bouwmeester, Harro J.

2004-01-01

The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as α-pinene and β-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of α-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of α-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species. PMID:15522848

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation.

PubMed

Sasaki, Daisuke; Fujihashi, Masahiro; Okuyama, Naomi; Kobayashi, Yukiko; Noike, Motoyoshi; Koyama, Tanetoshi; Miki, Kunio

2011-02-04

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.

A kinetic study on the chemical cleavage of nucleoside diphosphate sugars.

PubMed

Huhta, Eija; Parjanen, Atte; Mikkola, Satu

2010-03-30

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP. Copyright 2009 Elsevier Ltd. All rights reserved.

Monoterpenoid biosynthesis in Saccharomyces cerevisiae.

PubMed

Oswald, Marilyne; Fischer, Marc; Dirninger, Nicole; Karst, Francis

2007-05-01

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.

Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots.

PubMed

Youssef, Reham M; Kim, Kyung-Hwan; Haroon, Sanaa A; Matthews, Benjamin F

2013-06-01

Plant parasitic nematodes cause approximately 157 billion US dollars in losses worldwide annually. The soybean cyst nematode (SCN), Heterodera glycines, is responsible for an estimated one billion dollars in losses to the US farmer each year. A promising new approach for control of plant parasitic nematode control is gene silencing. We tested this approach by silencing the SCN gene HgALD, encoding fructose-1,6-diphosphate aldolase. This enzyme is important in the conversion of glucose into energy and may be especially important in actin-based motility during parasite invasion of its host. An RNAi construct targeted to silence HgALD was transformed into soybean roots of composite plants to examine its efficacy to reduce the development of females formed by SCN. The number of mature females on roots transformed with the RNAi construct designed to silence the HgALD gene was reduced by 58%. These results indicate that silencing the aldolase gene of SCN +can greatly decrease the number of female SCN reaching maturity, and it is a promising step towards broadening resistance of plants against plant-parasitic nematodes. Published by Elsevier Inc.

The compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells.

PubMed

Bettendorff, L

1994-05-26

Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier (KM = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [14C]thiamine or [14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1-2 h. When preloading the cells with [14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism.

Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

PubMed

Rhaese, H J; Hoch, J A; Groscurth, R

1977-03-01

To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Biosensor reveals multiple sources for mitochondrial NAD⁺.

PubMed

Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

2016-06-17

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

PubMed

Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

2011-06-01

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L.

PubMed

Jones, Christopher G; Keeling, Christopher I; Ghisalberti, Emilio L; Barbour, Elizabeth L; Plummer, Julie A; Bohlmann, Jörg

2008-09-01

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731bp) and SasesquiTPS1 (1680bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-alpha-terpineol and (-)-limonene, along with small quantities of linalool, myrcene, (-)-alpha-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were alpha-bulnesene, gamma-muurolene, alpha- and beta-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

PubMed Central

Hailat, N; Keim, D R; Melhem, R F; Zhu, X X; Eckerskorn, C; Brodeur, G M; Reynolds, C P; Seeger, R C; Lottspeich, F; Strahler, J R

1991-01-01

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors. Images PMID:2056128

Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production1

PubMed Central

Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi

2015-01-01

Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. PMID:26438788

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa

PubMed Central

Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

2010-01-01

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum. PMID:20421194

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

PubMed

Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

2010-06-01

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity▿

PubMed Central

Withers, Sydnor T.; Gottlieb, Shayin S.; Lieu, Bonny; Newman, Jack D.; Keasling, Jay D.

2007-01-01

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol. PMID:17693564

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

PubMed Central

Hoeffler, Jean-François; Hemmerlin, Andréa; Grosdemange-Billiard, Catherine; Bach, Thomas J; Rohmer, Michel

2002-01-01

In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-(2)H]deoxy-d-xylulose ([4-(2)H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-(2)H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase. PMID:12010124

New Synthetic Methodology for the Construction of 7-Substituted Farnesyl Diphosphate Analogs

PubMed Central

Placzek, Andrew T.

2012-01-01

Through the use of a 1,2-metalate rearrangement, six 7-substituted farnesol analogs were generated in a concise manner. This new synthetic route allowed us to quickly prepare several diverse farnesyl diphosphate analogs with interesting biological activities against mammalian protein-farnesyl transferase. PMID:21699139

Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids.

PubMed

Lan, Jian-bin; Yu, Rang-cai; Yu, Yun-yi; Fan, Yan-ping

2013-04-15

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.

Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis).

PubMed

Liu, Xiaogang; Lin, Cailing; Ma, Xiaodi; Tan, Yan; Wang, Jiuzhao; Zeng, Ming

2018-01-01

Fruits of sweet orange ( Citrus sinensis ), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O -glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O -rutinosides or O -neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7- O -rhamnoside, quercetin 7- O -glucoside, and kaempferol 7- O -glucoside, implying that the enzyme has flavonoid 7- O -glucosyltransferase and 7- O -rhamnosyltransferase activities in vivo .

Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis)

PubMed Central

Liu, Xiaogang; Lin, Cailing; Ma, Xiaodi; Tan, Yan; Wang, Jiuzhao; Zeng, Ming

2018-01-01

Fruits of sweet orange (Citrus sinensis), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O-glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O-rutinosides or O-neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7-O-rhamnoside, quercetin 7-O-glucoside, and kaempferol 7-O-glucoside, implying that the enzyme has flavonoid 7-O-glucosyltransferase and 7-O-rhamnosyltransferase activities in vivo. PMID:29497429

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases.

PubMed

Grabińska, Kariona A; Park, Eon Joo; Sessa, William C

2016-08-26

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. cis-PTs catalyze consecutive condensation reactions of allylic diphosphate acceptor with isopentenyl diphosphate (IPP) in the cis (Z) configuration to generate linear polyprenyl diphosphate. The chain lengths of isoprenoid carbon skeletons vary widely from neryl pyrophosphate (C10) to natural rubber (C>10,000). The homo-dimeric bacterial enzyme, undecaprenyl diphosphate synthase (UPPS), has been structurally and mechanistically characterized in great detail and serves as a model for understanding the mode of action of eukaryotic cis-PTs. However, recent experiments have revealed that mammals, fungal, and long-chain plant cis-PTs are heteromeric enzymes composed of two distantly related subunits. In this review, the classification, function, and evolution of cis-PTs will be discussed with a special emphasis on the role of the newly described NgBR/Nus1 subunit and its plants' orthologs as essential, structural components of the cis-PTs activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Bacopa monniera recombinant mevalonate diphosphate decarboxylase: Biochemical characterization.

PubMed

Abbassi, Shakeel J; Vishwakarma, Rishi K; Patel, Parth; Kumari, Uma; Khan, Bashir M

2015-08-01

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 μM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+). Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production.

PubMed

Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi; Wong, Limsoon; Chua, Nam-Hai; Jang, In-Cheol

2015-12-01

Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. © 2015 American Society of Plant Biologists. All Rights Reserved.

Identification of a novel gene cluster participating in menaquinone (vitamin K2) biosynthesis. Cloning and sequence determination of the 2-heptaprenyl-1,4-naphthoquinone methyltransferase gene of Bacillus stearothermophilus.

PubMed

Koike-Takeshita, A; Koyama, T; Ogura, K

1997-05-09

We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, which catalyzes the synthesis of the prenyl side chain of menaquinone-7 of this bacterium. Sequence analyses revealed the presence of three open reading frames (ORFs), designated as ORF-1, ORF-2, and ORF-3, and the structural genes of the heptaprenyl diphosphate synthase were proved to consist of ORF-1 (heps-1) and ORF-3 (heps-2) (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J. Biol. Chem. 270, 18396-18400). The predicted amino acid sequence of ORF-2 (234 amino acids) contains a methyltransferase consensus sequence and shows a 22% identity with UbiG of Escherichia coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free homogenate of the transformant of E. coli with an expression vector of ORF-2 catalyzed the incorporation of S-adenosyl-L-methionine into menaquinone-8, indicating that ORF-2 encodes 2-heptaprenyl-1,4-naphthoquinone methyltransferase, which participates in the terminal step of the menaquinone biosynthesis. Thus it is concluded that the ORF-1, ORF-2, and ORF-3 genes, designated heps-1, menG, and heps-2, respectively, form another cluster involved in menaquinone biosynthesis in addition to the cluster of menB, menC, menD, and menE already identified in the Bacillus subtilis and E. coli chromosomes.

The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish.

PubMed

Busch-Nentwich, Elisabeth; Söllner, Christian; Roehl, Henry; Nicolson, Teresa

2004-02-01

Over 30 genes responsible for human hereditary hearing loss have been identified during the last 10 years. The proteins encoded by these genes play roles in a diverse set of cellular functions ranging from transcriptional regulation to K(+) recycling. In a few cases, the genes are novel and do not give much insight into the cellular or molecular cause for the hearing loss. Among these poorly understood deafness genes is DFNA5. How the truncation of the encoded protein DFNA5 leads to an autosomal dominant form of hearing loss is not clear. In order to understand the biological role of Dfna5, we took a reversegenetic approach in zebrafish. Here we show that morpholino antisense nucleotide knock-down of dfna5 function in zebrafish leads to disorganization of the developing semicircular canals and reduction of pharyngeal cartilage. This phenotype closely resembles previously isolated zebrafish craniofacial mutants including the mutant jekyll. jekyll encodes Ugdh [uridine 5'-diphosphate (UDP)-glucose dehydrogenase], an enzyme that is crucial for production of the extracellular matrix component hyaluronic acid (HA). In dfna5 morphants, expression of ugdh is absent in the developing ear and pharyngeal arches, and HA levels are strongly reduced in the outgrowing protrusions of the developing semicircular canals. Previous studies suggest that HA is essential for differentiating cartilage and directed outgrowth of the epithelial protrusions in the developing ear. We hypothesize that the reduction of HA production leads to uncoordinated outgrowth of the canal columns and impaired facial cartilage differentiation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.

The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. Thesemore » structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.« less

Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production.

PubMed

Chaves, Julie E; Romero, Paloma Rueda; Kirst, Henning; Melis, Anastasios

2016-12-01

Heterologous production of isoprene (C 5 H 8 ) hydrocarbons in cyanobacteria, emanating from sunlight, CO 2 , and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Dowd, Bing; Williams, Sarah; Wang, Hongxin

Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target described is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H + /2e - reduction. IspH contains a 4 Fe-4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe-4 S clusters by η 1 coordination, forming tetrahedral geometries at themore » unique fourth Fe, ligand side chains preventing further ligand (e.g., H 2 O, O 2 ) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a K i value of ≈500 nm against Pseudomonas aeruginosa IspH.« less

Elevated guanosine 5'-diphosphate 3'-diphosphate level inhibits bacterial growth and interferes with FtsZ assembly.

PubMed

Yamaguchi, Takayoshi; Iida, Ken-Ichiro; Shiota, Susumu; Nakayama, Hiroaki; Yoshida, Shin-Ichi

2015-12-01

FtsZ, a protein essential for prokaryotic cell division, forms a ring structure known as the Z-ring at the division site. FtsZ has a GTP binding site and is assembled into linear structures in a GTP-dependent manner in vitro. We assessed whether guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a global regulator of gene expression in starved bacteria, affects cell division in Salmonella Paratyphi A. Elevation of intracellular ppGpp levels by using the relA expression vector induced repression of bacterial growth and incorrect FtsZ assembly. We found that FtsZ forms helical structures in the presence of ppGpp by using the GTP binding site; however, ppGpp levels required to form helical structures were at least 20-fold higher than the required GTP levels in vitro. Furthermore, once formed, helical structures did not change to the straight form even after GTP addition. Our data indicate that elevation of the ppGpp level leads to inhibition of bacterial growth and interferes with FtsZ assembly. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inhibitory effect of phosphates on magnesium lactate efflorescence formation in dry-fermented sausages.

PubMed

Walz, Felix H; Gibis, Monika; Schrey, Pia; Herrmann, Kurt; Reichert, Corina L; Hinrichs, Jörg; Weiss, Jochen

2017-10-01

This study aimed to prevent the phenomena of efflorescence formation on the surface of dry fermented sausages due to the complexation of efflorescence forming cations with phosphates. Efflorescence formation is a critical issue constituting a major quality defect, especially of dry fermented sausages. Different phosphates (di- and hexametaphosphate) were added (3.0g/kg) to the sausage batter. As a hypothesis, these additives should complex with one of the main efflorescence-causing substances such as magnesium. The formation of efflorescences was determined for dry fermented sausages without phosphate addition, with diphosphate, or hexametaphosphate addition during 8weeks of storage under modified atmosphere. The visual analyses of the sausage surface revealed high amounts of efflorescences for the control (42.2%) and for the sausages with added diphosphate (40.9%), whereas the sausages containing hexametaphosphate had significantly reduced amounts of efflorescence formation, showing only 11.9% efflorescences after 8weeks of storage. This inhibition was a result of strong complexation of hexametaphosphate with magnesium ions, thus preventing the diffusion of magnesium towards the sausage surface. This can be explained by the magnesium content on the sausage surface that increased by 163.9, 127.8, and 52.8% for the sausages without phosphate, diphosphate, and hexametaphosphate addition, respectively. The mass transport of lactate and creatine was not affected by phosphate addition. Isothermal titration calorimetry confirmed that, theoretically, 4.5g/kg of diphosphate or 2.8g/kg hexametaphosphate are required to complex 0.2g/kg magnesium ions naturally occurring in dry fermented sausages and, thus, the chosen overall phosphate concentration of 3.0g/kg was enough when adding hexametaphosphate, but not for diphosphate, to inhibit the efflorescence formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

PubMed

Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.

PubMed

Dowdle, John; Ishikawa, Takahiro; Gatzek, Stephan; Rolinski, Susanne; Smirnoff, Nicholas

2007-11-01

Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.

Culture temperature affects gene expression and metabolic pathways in the 2-methylisoborneol-producing cyanobacterium Pseudanabaena galeata.

PubMed

Kakimoto, Masayuki; Ishikawa, Toshiki; Miyagi, Atsuko; Saito, Kazuaki; Miyazaki, Motonobu; Asaeda, Takashi; Yamaguchi, Masatoshi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2014-02-15

A volatile metabolite, 2-methylisoborneol (2-MIB), causes an unpleasant taste and odor in tap water. Some filamentous cyanobacteria produce 2-MIB via a two-step biosynthetic pathway: methylation of geranyl diphosphate (GPP) by methyl transferase (GPPMT), followed by the cyclization of methyl-GPP by monoterpene cyclase (MIBS). We isolated the genes encoding GPPMT and MIBS from Pseudanabaena galeata, a filamentous cyanobacterium known to be a major causal organism of 2-MIB production in Japanese lakes. The predicted amino acid sequence showed high similarity with that of Pseudanabaena limnetica (96% identity in GPPMT and 97% identity in MIBS). P. galeata was cultured at different temperatures to examine the effect of growth conditions on the production of 2-MIB and major metabolites. Gas chromatograph-mass spectrometry (GC-MS) measurements showed higher accumulation of 2-MIB at 30 °C than at 4 °C or 20 °C after 24 h of culture. Real-time-RT PCR analysis showed that the expression levels of the genes encoding GPPMT and MIBS decreased at 4 °C and increased at 30 °C, compared with at 20 °C. Furthermore, metabolite analysis showed dramatic changes in primary metabolite concentrations in cyanobacteria grown at different temperatures. The data indicate that changes in carbon flow in the TCA cycle affect 2-MIB biosynthesis at higher temperatures. Copyright © 2013 Elsevier GmbH. All rights reserved.

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

PubMed

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

PubMed Central

Van Moerkercke, Alex; Steensma, Priscille; Schweizer, Fabian; Pollier, Jacob; Gariboldi, Ivo; Payne, Richard; Vanden Bossche, Robin; Miettinen, Karel; Espoz, Javiera; Purnama, Purin Candra; Kellner, Franziska; Seppänen-Laakso, Tuulikki; O’Connor, Sarah E.; Rischer, Heiko; Memelink, Johan; Goossens, Alain

2015-01-01

Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix–loop–helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures. PMID:26080427

Transcriptomic analysis reveals numerous diverse protein kinases and transcription factors involved in desiccation tolerance in the resurrection plant Myrothamnus flabellifolia

PubMed Central

Ma, Chao; Wang, Hong; Macnish, Andrew J; Estrada-Melo, Alejandro C; Lin, Jing; Chang, Youhong; Reid, Michael S; Jiang, Cai-Zhong

2015-01-01

The woody resurrection plant Myrothamnus flabellifolia has remarkable tolerance to desiccation. Pyro-sequencing technology permitted us to analyze the transcriptome of M. flabellifolia during both dehydration and rehydration. We identified a total of 8287 and 8542 differentially transcribed genes during dehydration and rehydration treatments respectively. Approximately 295 transcription factors (TFs) and 484 protein kinases (PKs) were up- or down-regulated in response to desiccation stress. Among these, the transcript levels of 53 TFs and 91 PKs increased rapidly and peaked early during dehydration. These regulators transduce signal cascades of molecular pathways, including the up-regulation of ABA-dependent and independent drought stress pathways and the activation of protective mechanisms for coping with oxidative damage. Antioxidant systems are up-regulated, and the photosynthetic system is modified to reduce ROS generation. Secondary metabolism may participate in the desiccation tolerance of M. flabellifolia as indicated by increases in transcript abundance of genes involved in isopentenyl diphosphate biosynthesis. Up-regulation of genes encoding late embryogenesis abundant proteins and sucrose phosphate synthase is also associated with increased tolerance to desiccation. During rehydration, the transcriptome is also enriched in transcripts of genes encoding TFs and PKs, as well as genes involved in photosynthesis, and protein synthesis. The data reported here contribute comprehensive insights into the molecular mechanisms of desiccation tolerance in M. flabellifolia. PMID:26504577

Structural characterization of alpha-terminal group of natural rubber. 1. Decomposition of branch-points by lipase and phosphatase treatments.

PubMed

Tarachiwin, Lucksanaporn; Sakdapipanich, Jitladda; Ute, Koichi; Kitayama, Tatsuki; Bamba, Takashi; Fukusaki, Ei-Ichiro; Kobayashi, Akio; Tanaka, Yasuyuki

2005-01-01

Deproteinized natural rubber latex (DPNR-latex) was treated with lipase and phosphatase in order to analyze the structure of the chain-end group (alpha-terminal). The enzymatic treatment decreased the content of long-chain fatty acid ester groups in DPNR from about 6 to 2 mol per rubber molecule. The molecular weight and intrinsic viscosity were reduced to about one-third after treatment with lipase and phosphatase. The Huggins' k' constant of the enzyme-treated DPNR showed the formation of linear rubber molecules. The molecular weight distribution of DPNR changed apparently after treatment with lipase and phosphatase. (1)H NMR spectrum of rubber obtained from DPNR-latex showed small signals due to monophosphate, di-phosphate and phospholipids at the alpha-terminus. Treatment of DPNR-latex with lipase and phosphatase decreased the relative intensity of the (1)H NMR signals corresponding to phospholipids, whereas no change was observed for the signals due to mono- and diphosphates. The residual mono- and diphosphate signals as well as some phospholipid signals after lipase and phosphatase treatments indicate that mono- and diphosphate groups are directly linked at the alpha-terminus with the modified structure, expected by aggregation or linking with phospholipid molecules.

Optimization of primaquine diphosphate tablet formulation for controlled drug release using the mixture experimental design.

PubMed

Duque, Marcelo Dutra; Kreidel, Rogério Nepomuceno; Taqueda, Maria Elena Santos; Baby, André Rolim; Kaneko, Telma Mary; Velasco, Maria Valéria Robles; Consiglieri, Vladi Olga

2013-01-01

A tablet formulation based on hydrophilic matrix with a controlled drug release was developed, and the effect of polymer concentrations on the release of primaquine diphosphate was evaluated. To achieve this purpose, a 20-run, four-factor with multiple constraints on the proportions of the components was employed to obtain tablet compositions. Drug release was determined by an in vitro dissolution study in phosphate buffer solution at pH 6.8. The polynomial fitted functions described the behavior of the mixture on simplex coordinate systems to study the effects of each factor (polymer) on tablet characteristics. Based on the response surface methodology, a tablet composition was optimized with the purpose of obtaining a primaquine diphosphate release closer to a zero order kinetic. This formulation released 85.22% of the drug for 8 h and its kinetic was studied regarding to Korsmeyer-Peppas model, (Adj-R(2) = 0.99295) which has confirmed that both diffusion and erosion were related to the mechanism of the drug release. The data from the optimized formulation were very close to the predictions from statistical analysis, demonstrating that mixture experimental design could be used to optimize primaquine diphosphate dissolution from hidroxypropylmethyl cellulose and polyethylene glycol matrix tablets.

Investigations of systems ThO 2-MO 2-P 2O 5 (M=U, Ce, Zr, Pu). Solid solutions of thorium-uranium (IV) and thorium-plutonium (IV) phosphate-diphosphates

NASA Astrophysics Data System (ADS)

Dacheux, N.; Podor, R.; Brandel, V.; Genet, M.

1998-02-01

In the framework of nuclear waste management aiming at the research of a storage matrix, the chemistry of thorium phosphates has been completely re-examined. In the ThO 2-P 2O 5 system a new compound thorium phosphate-diphosphate Th 4(PO 4) 4P 2O 7 has been synthesized. The replacement of Th 4+ by a smaller cation like U 4+ and Pu 4+ in the thorium phosphate-diphosphate (TPD) lattice has been achieved. Th 4- xU x(PO 4) 4P 2O 7 and Th 4- xPu x(PO 4) 4P 2O 7 solid solutions have been synthesized through wet and dry processes with 0< x<3.0 for uranium and 0< x<1.0 for plutonium. From the variation of the unit cell parameters, an upper x value equal to 1.67 has been estimated for the thorium-plutonium (IV) phosphate-diphosphate solid solutions. Two other tetravalent cations, Ce 4+ and Zr 4+, cannot be incorporated in the TPD lattice: cerium (IV) because of its reduction into Ce (III) at high temperature, and zirconium probably because of its too small radius compared to thorium.

The Variability of Sesquiterpenes Emitted from Two Zea mays Cultivars Is Controlled by Allelic Variation of Two Terpene Synthase Genes Encoding Stereoselective Multiple Product Enzymes

PubMed Central

Köllner, Tobias G.; Schnee, Christiane; Gershenzon, Jonathan; Degenhardt, Jörg

2004-01-01

The mature leaves and husks of Zea mays release a complex blend of terpene volatiles after anthesis consisting predominantly of bisabolane-, sesquithujane-, and bergamotane-type sesquiterpenes. The varieties B73 and Delprim release the same volatile constituents but in significantly different proportions. To study the molecular genetic and biochemical mechanisms controlling terpene diversity and distribution in these varieties, we isolated the closely related terpene synthase genes terpene synthase4 (tps4) and tps5 from both varieties. The encoded enzymes, TPS4 and TPS5, each formed the same complex mixture of sesquiterpenes from the precursor farnesyl diphosphate but with different proportions of products. These mixtures correspond to the sesquiterpene blends observed in the varieties B73 and Delprim, respectively. The differences in the stereoselectivity of TPS4 and TPS5 are determined by four amino acid substitutions with the most important being a Gly instead of an Ala residue at position 409 at the catalytic site of the enzyme. Although both varieties contain tps4 and tps5 alleles, their differences in terpene composition result from the fact that B73 has only a single functional allele of tps4 and no functional alleles of tps5, whereas Delprim has only a functional allele of tps5 and no functional alleles of tps4. Lack of functionality was shown to be attributable to frame-shift mutations or amino acid substitutions that greatly reduce the activity of their encoded proteins. Therefore, the diversity of sesquiterpenes in these two maize cultivars is strongly influenced by single nucleotide changes in the alleles of two terpene synthase genes. PMID:15075399

The Maize Gene terpene synthase 1 Encodes a Sesquiterpene Synthase Catalyzing the Formation of (E)-β-Farnesene, (E)-Nerolidol, and (E,E)-Farnesol after Herbivore Damage1

PubMed Central

Schnee, Christiane; Köllner, Tobias G.; Gershenzon, Jonathan; Degenhardt, Jörg

2002-01-01

Maize (Zea mays) emits a mixture of volatile compounds upon attack by the Egyptian cotton leafworm (Spodoptera littoralis). These substances, primarily mono- and sesquiterpenes, are used by parasitic wasps to locate the lepidopteran larvae, which are their natural hosts. This interaction among plant, lepidopteran larvae, and hymenopteran parasitoids benefits the plant and has been termed indirect defense. The committed step in the biosynthesis of the different skeletal types of mono- and sesquiterpenes is catalyzed by terpene synthases, a class of enzymes that forms a large variety of mono- and sesquiterpene products from prenyl diphosphate precursors. We isolated a terpene synthase gene, terpene synthase 1 (tps1), from maize that exhibits only a low degree of sequence identity to previously identified terpene synthases. Upon expression in a bacterial system, the encoded enzyme produced the acyclic sesquiterpenes, (E)-β-farnesene, (E,E)-farnesol, and (3R)-(E)-nerolidol, the last an intermediate in the formation of (3E)-4,8-dimethyl-1,3,7-nonatriene. Both (E)-β-farnesene and (3E)-4,8-dimethyl-1,3,7-nonatriene are prominent compounds of the maize volatile blend that is emitted after herbivore damage. The biochemical characteristics of the encoded enzyme are similar to those of terpene synthases from both gymnosperms and dicotyledonous angiosperms, suggesting that catalysis involves a similar electrophilic reaction mechanism. The transcript level of tps1 in the maize cv B73 was elevated after herbivory, mechanical damage, and treatment with elicitors. In contrast, the increase in the transcript level of the tps1 gene or gene homolog in the maize cv Delprim after herbivory was less pronounced, suggesting that the regulation of terpene synthase expression may vary among maize varieties. PMID:12481088

The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes.

PubMed

Köllner, Tobias G; Schnee, Christiane; Gershenzon, Jonathan; Degenhardt, Jörg

2004-05-01

The mature leaves and husks of Zea mays release a complex blend of terpene volatiles after anthesis consisting predominantly of bisabolane-, sesquithujane-, and bergamotane-type sesquiterpenes. The varieties B73 and Delprim release the same volatile constituents but in significantly different proportions. To study the molecular genetic and biochemical mechanisms controlling terpene diversity and distribution in these varieties, we isolated the closely related terpene synthase genes terpene synthase4 (tps4) and tps5 from both varieties. The encoded enzymes, TPS4 and TPS5, each formed the same complex mixture of sesquiterpenes from the precursor farnesyl diphosphate but with different proportions of products. These mixtures correspond to the sesquiterpene blends observed in the varieties B73 and Delprim, respectively. The differences in the stereoselectivity of TPS4 and TPS5 are determined by four amino acid substitutions with the most important being a Gly instead of an Ala residue at position 409 at the catalytic site of the enzyme. Although both varieties contain tps4 and tps5 alleles, their differences in terpene composition result from the fact that B73 has only a single functional allele of tps4 and no functional alleles of tps5, whereas Delprim has only a functional allele of tps5 and no functional alleles of tps4. Lack of functionality was shown to be attributable to frame-shift mutations or amino acid substitutions that greatly reduce the activity of their encoded proteins. Therefore, the diversity of sesquiterpenes in these two maize cultivars is strongly influenced by single nucleotide changes in the alleles of two terpene synthase genes.

Enhanced Production of Polysaccharide Through the Overexpression of Homologous Uridine Diphosphate Glucose Pyrophosphorylase Gene in a Submerged Culture of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes).

PubMed

Ji, Sen-Lin; Liu, Rui; Ren, Meng-Fei; Li, Huan-Jun; Xu, Jun-Wei

2015-01-01

This study aimed to improve polysaccharide production by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of the homologous UDP glucose pyrophosphorylase (UGP) gene. The effects of UGP gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production, and transcription levels of 3 genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), UGP, and α-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the UGP gene were 24.32 mg/100 mg dry weight and 1.66 g/L, respectively, which were higher by 42% and 36% than those of the wild-type strain. The transcription levels of PGM, UGP, and GLS were up-regulated by 1.6, 2.6, and 2.4-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

PubMed Central

Carta, Claudio; Pantaleoni, Francesca; Bocchinfuso, Gianfranco; Stella, Lorenzo; Vasta, Isabella; Sarkozy, Anna; Digilio, Cristina; Palleschi, Antonio; Pizzuti, Antonio; Grammatico, Paola; Zampino, Giuseppe; Dallapiccola, Bruno; Gelb, Bruce D.; Tartaglia, Marco

2006-01-01

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring–binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor. PMID:16773572

Mutations in PCYT1A cause spondylometaphyseal dysplasia with cone-rod dystrophy.

PubMed

Yamamoto, Guilherme L; Baratela, Wagner A R; Almeida, Tatiana F; Lazar, Monize; Afonso, Clara L; Oyamada, Maria K; Suzuki, Lisa; Oliveira, Luiz A N; Ramos, Ester S; Kim, Chong A; Passos-Bueno, Maria Rita; Bertola, Débora R

2014-01-02

Spondylometaphyseal dysplasia with cone-rod dystrophy is a rare autosomal-recessive disorder characterized by severe short stature, progressive lower-limb bowing, flattened vertebral bodies, metaphyseal involvement, and visual impairment caused by cone-rod dystrophy. Whole-exome sequencing of four individuals affected by this disorder from two Brazilian families identified two previously unreported homozygous mutations in PCYT1A. This gene encodes the alpha isoform of the phosphate cytidylyltransferase 1 choline enzyme, which is responsible for converting phosphocholine into cytidine diphosphate-choline, a key intermediate step in the phosphatidylcholine biosynthesis pathway. A different enzymatic defect in this pathway has been previously associated with a muscular dystrophy with mitochondrial structural abnormalities that does not have cartilage and/or bone or retinal involvement. Thus, the deregulation of the phosphatidylcholine pathway may play a role in multiple genetic diseases in humans, and further studies are necessary to uncover its precise pathogenic mechanisms and the entirety of its phenotypic spectrum. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea

PubMed Central

Siewers, Verena; Smedsgaard, Jørn; Tudzynski, Paul

2004-01-01

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this “direct” pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: ΔBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene. PMID:15240257

Mutation K42E in dehydrodolichol diphosphate synthase (DHDDS) causes recessive retinitis pigmentosa.

PubMed

Lam, Byron L; Züchner, Stephan L; Dallman, Julia; Wen, Rong; Alfonso, Eduardo C; Vance, Jeffery M; Peričak-Vance, Margaret A

2014-01-01

A single-nucleotide mutation in the gene that encodes DHDDS has been identified by whole exome sequencing as the cause of the non-syndromic recessive retinitis pigmentosa (RP) in a family of Ashkenazi Jewish origin in which three of the four siblings have early onset retinal degeneration. The peripheral retinal degeneration in the affected siblings was evident in the initial examination in 1992 and only one had detectable electroretinogram (ERG) that suggested cone-rod dysfunction. The pigmentary retinal degeneration subsequently progressed rapidly. The identified mutation changes the highly conserved residue Lys42 to Glu, resulting in lower catalytic efficiency. Patterns of plasma transferrin isoelectric focusing gel were normal in all family members, indicating no significant abnormality in protein glycosylation. Dolichols have been shown to influence the fluidity and of the membrane and promote vesicle fusion. Considering that photoreceptor outer segments contain stacks of membrane discs, we believe that the mutation may lead to low dolichol levels in photoreceptor outer segments, resulting in unstable membrane structure that leads to photoreceptor degeneration.

Molecular and biochemical characterization of tomato farnesyl-protein transferase.

PubMed

Schmitt, D; Callan, K; Gruissem, W

1996-10-01

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.

Lovastatin inhibits amyloid precursor protein (APP) beta-cleavage through reduction of APP distribution in Lubrol WX extractable low density lipid rafts.

PubMed

Won, Je-Seong; Im, Yeong-Bin; Khan, Mushfiquddin; Contreras, Miguel; Singh, Avtar K; Singh, Inderjit

2008-05-01

Previous studies have described that statins (inhibitors of cholesterol and isoprenoid biosynthesis) inhibit the output of amyloid-beta (Abeta) in the animal model and thus decrease risk of Alzheimer's disease. However, their action mechanism(s) in Abeta precursor protein (APP) processing and Abeta generation is not fully understood. In this study, we report that lovastatin treatment reduced Abeta output in cultured hippocampal neurons as a result of reduced APP levels and beta-secretase activities in low density Lubrol WX (non-ionic detergent) extractable lipid rafts (LDLR). Rather than altering cholesterol levels in lipid raft fractions and thus disrupting lipid raft structure, lovastatin decreased Abeta generation through down-regulating geranylgeranyl-pyrophosphate dependent endocytosis pathway. The inhibition of APP endocytosis by treatment with lovastatin and reduction of APP levels in LDLR fractions by treatment with phenylarsine oxide (a general endocytosis inhibitor) support the involvement of APP endocytosis in APP distribution in LDLR fractions and subsequent APP beta-cleavage. Moreover, lovastatin-mediated down-regulation of endocytosis regulators, such as early endosomal antigen 1, dynamin-1, and phosphatidylinositol 3-kinase activity, indicates that lovastatin modulates APP endocytosis possibly through its pleiotropic effects on endocytic regulators. Collectively, these data report that lovastatin mediates inhibition of LDLR distribution and beta-cleavage of APP in a geranylgeranyl-pyrophosphate and endocytosis-dependent manner.

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR 343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR 343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plantmore » hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR 343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR 343.« less

A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization

PubMed Central

Bible, Amber N.; Fletcher, Sarah J.; Pelletier, Dale A.; Schadt, Christopher W.; Jawdy, Sara S.; Weston, David J.; Engle, Nancy L.; Tschaplinski, Timothy; Masyuko, Rachel; Polisetti, Sneha; Bohn, Paul W.; Coutinho, Teresa A.; Doktycz, Mitchel J.; Morrell-Falvey, Jennifer L.

2016-01-01

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343. PMID:27148182

A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization

DOE PAGES

Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A; ...

2016-04-18

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR 343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR 343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plantmore » hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR 343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR 343.« less

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

NASA Astrophysics Data System (ADS)

Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

A conserved C-terminal RXG motif in the NgBR subunit of cis-prenyltransferase is critical for prenyltransferase activity.

PubMed

Grabińska, Kariona A; Edani, Ban H; Park, Eon Joo; Kraehling, Jan R; Sessa, William C

2017-10-20

cis -Prenyltransferases ( cis -PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, cis -PT is the first enzyme committed to the synthesis of dolichyl phosphate, an obligate lipid carrier in protein glycosylation reactions. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase, generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently, we discovered that unlike undecaprenyl diphosphate synthase, mammalian cis -PT is a heteromer consisting of NgBR (Nus1) and hCIT (dehydrodolichol diphosphate synthase) subunits, and this composition has been confirmed in plants and fungal cis -PTs. Here, we establish the first purification system for heteromeric cis -PT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical R X G sequence in the C-terminal tail of NgBR that is conserved and essential for enzyme activity across phyla. In summary, our findings show that eukaryotic cis -PT is composed of the NgBR and hCIT subunits. The strong conservation of the R X G motif among NgBR orthologs indicates that this subunit is critical for the synthesis of polyprenol diphosphates and cellular function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Strength Characteristics of Resorbable Osteoconductive Ceramics Based on Diphosphates of Calcium and Alkali Metals

NASA Astrophysics Data System (ADS)

Putlayev, V. I.; Evdokimov, P. V.; Garshev, A. V.; Prosvirin, D. V.; Klimashina, E. S.; Safronova, T. V.; Ivanov, V. K.

2014-02-01

An investigation into the strength characteristics of ceramics based on diphosphates Ca(3- x)М2 x (PO4)2 ( x = 0-1 and М = Na, K) provides evidence of composition strengthening in the range х = 0.6-0.8 containing the greatest amount of the supercooled high-temperature modification α-СаМРО4. The method of high-temperature x-ray diffractometry is used to examine thermal expansion of rhenanite phases of СаМРО4.

Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

PubMed Central

Yang, Jiangang; Li, Jitao; Men, Yan; Zhu, Yueming; Zhang, Ying; Ma, Yanhe

2015-01-01

The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars. PMID:25888171

Unusual features of a recombinant apple alpha-farnesene synthase.

PubMed

Green, Sol; Friel, Ellen N; Matich, Adam; Beuning, Lesley L; Cooney, Janine M; Rowan, Daryl D; MacRae, Elspeth

2007-01-01

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.

Effect of long-term clopidogrel treatment on platelet function and inflammation in patients undergoing coronary arterial stenting.

PubMed

Antonino, Mark J; Mahla, Elisabeth; Bliden, Kevin P; Tantry, Udaya S; Gurbel, Paul A

2009-06-01

A clopidogrel loading dose administered during stenting attenuates inflammation marker release. However, less is known of the anti-inflammatory effect of clopidogrel maintenance therapy. Platelet reactivity to adenosine diphosphate and inflammation markers were measured in 110 consecutive patients (69 clopidogrel-naive patients and 41 patients receiving long-term clopidogrel therapy for >6 months) before nonemergent stenting by turbidimetric aggregometry and flow cytometry and multianalyte profiling, respectively. All patients were treated with aspirin. Prestenting adenosine diphosphate-induced platelet aggregation, P-selectin, and activated glycoprotein IIb/IIIa expression were lower in patients receiving long-term clopidogrel therapy compared with the clopidogrel-naive group (p <0.001), accompanied by lower levels of selected inflammation markers (p < or = 0.05). Additionally, there were strong correlations between platelet aggregation and flow cytometric measurements (p < or = 0.04) and between specific inflammation markers (p < or = 0.02). In conclusion, in addition to markedly lowering platelet reactivity to adenosine diphosphate, long-term clopidogrel therapy is associated with an anti-inflammatory effect.

Silver indium diphosphate, AgInP(2)O(7).

PubMed

Zouihri, Hafid; Saadi, Mohamed; Jaber, Boujemaa; El Ammari, Lehcen

2010-12-18

Polycrystalline material of the title compound, AgInP(2)O(7), was synthesized by traditional high-temperature solid-state methods and single crystals were grown from the melt of a mixture of AgInP(2)O(7) and B(2)O(3) as flux in a platinium crucible. The structure consists of InO(6) octa-hedra, which are corner-shared to PO(4) tetra-hedra into a three-dimensional network with hexa-gonal channels running parallel to the c axis. The silver cation, located in the channel, is bonded to seven O atoms of the [InP(2)O(7)] framework with Ag-O distances ranging from 2.370 (2) to 3.015 (2) Å. The P(2)O(7) diphosphate anion is characterized by a P-O-P angle of 137.27 (9) and a nearly eclipsed conformation. AgInP(2)O(7) is isotypic with the M(I)FeP(2)O(7) (M(I) = Na, K, Rb, Cs and Ag) diphosphate family.

Biosynthetic elongation of isolated teichuronic acid polymers via glucosyl- and N-acetylmannosaminuronosyltransferases from solubilized cytoplasmic membrane fragments of Micrococcus luteus.

PubMed Central

Hildebrandt, K M; Anderson, J S

1990-01-01

Cytoplasmic membrane fragments of Micrococcus luteus catalyze in vitro biosynthesis of teichuronic acid from uridine diphosphate D-glucose (UDP-glucose), uridine diphosphate N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), and uridine diphosphate N-acetyl-D-glucosamine. Membrane fragments solubilized with Thesit (dodecyl alcohol polyoxyethylene ether) can utilize UDP-glucose and UDP-ManNAcA to effect elongation of teichuronic acid isolated from native cell walls. When UDP-glucose is the only substrate supplied, the detergent-solubilized glucosyltransferase incorporates a single glucosyl residue onto each teichuronic acid acceptor. When both UDP-glucose and UDP-ManNAcA are supplied, the glucosyltransferase and the N-acetylmannosaminuronosyltransferase act cooperatively to elongate the teichuronic acid acceptor by multiple additions of the disaccharide repeat unit. As shown by polyacrylamide gel electrophoresis, low-molecular-weight fractions of teichuronic acid are converted to higher-molecular-weight polymers by the addition of as many as 17 disaccharide repeat units. Images PMID:2118507

Probing the transcriptome of Aconitum carmichaelii reveals the candidate genes associated with the biosynthesis of the toxic aconitine-type C19-diterpenoid alkaloids.

PubMed

Zhao, Dake; Shen, Yong; Shi, Yana; Shi, Xingqiao; Qiao, Qin; Zi, Shuhui; Zhao, Erqiang; Yu, Diqiu; Kennelly, Edward J

2018-05-11

Aconitum carmichaelii has long been used as a traditional Chinese medicine, and its processed lateral roots are known commonly as fuzi. Aconitine-type C 19 -diterpenoid alkaloids accumulating in the lateral roots are some of the main toxicants of this species, yet their biosynthesis remains largely unresolved. As a first step towards understanding the biosynthesis of aconitine-type C 19 -diterpenoid alkaloids, we performed de novo transcriptome assembly and analysis of rootstocks and leaf tissues of Aconitum carmichaelii by next-generation sequencing. A total of 525 unigene candidates were identified as involved in the formation of C 19 -diterpenoid alkaloids, including those encoding enzymes in the early steps of diterpenoid alkaloids scaffold biosynthetic pathway, such as ent-copalyl diphosphate synthases, ent-kaurene synthases, kaurene oxidases, cyclases, and key aminotransferases. Furthermore, candidates responsible for decorating of diterpenoid alkaloid skeletons were discovered from transcriptome sequencing of fuzi, such as monooxygenases, methyltransferase, and BAHD acyltransferases. In addition, 645 differentially expressed genes encoding transcription factors potentially related to diterpenoid alkaloids accumulation underground were documented. Subsequent modular domain structure phylogenetics and differential expression analysis led to the identification of BAHD acyltransferases possibly involved in the formation of acetyl and benzoyl esters of diterpenoid alkaloids, associated with the acute toxicity of fuzi. The transcriptome data provide the foundation for future research into the molecular basis for aconitine-type C 19 -diterpenoid alkaloids biosynthesis in A. carmichaelii. Copyright © 2018. Published by Elsevier Ltd.

Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli.

PubMed Central

Gaal, T; Gourse, R L

1990-01-01

rRNA synthesis in Escherichia coli is subject to at least two regulation systems, growth rate-dependent control and stringent control. The inverse correlation between rRNA synthesis rates and guanosine 3'-diphosphate 5'-diphosphate (ppGpp) levels under various physiological conditions has led to the supposition that ppGpp is the mediator of both control mechanisms by inhibiting transcription from rrn P1 promoters. Recently, relA- spoT- strains have been constructed in which both ppGpp synthesis pathways most likely have been removed (M. Cashel, personal communication). We have confirmed that such strains produce no detectable ppGpp and therefore offer a direct means for testing the involvement of ppGpp in the regulation of rRNA synthesis in vivo. Stringent control was determined by measurement of rRNA synthesis after amino acid starvation, while growth rate control was determined by measurement of rRNA synthesis under different nutritional conditions. As expected, the relA- spoT- strain is relaxed for stringent control. However, growth rate-dependent regulation is unimpaired. These results indicate that growth rate regulation can occur in the absence of ppGpp and imply that ppGpp is not the mediator, or at least is not the sole mediator, of growth rate-dependent control. Therefore, growth rate-dependent control and stringent control may utilize different mechanisms for regulating stable RNA synthesis. PMID:2196571

Synthesis of the coenzymes adenosine diphosphate glucose, guanosine diphosphate glucose, and cytidine diphosphoethanolamine under primitive Earth conditions

NASA Technical Reports Server (NTRS)

Mar, A.; Oro, J.

1991-01-01

The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.

Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon

PubMed Central

2010-01-01

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5′-phosphosulfate or 3′,5′-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2′,3′-cylic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-l-arabinofuranose, guanosine 5'-diphosphate (GDP)-l-galactofuranose, UDP-l-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches. PMID:21043458

Reactions of fac-[Re(CO)3(H2O)3]+ with nucleoside diphosphates and thiamine diphosphate in aqueous solution investigated by multinuclear NMR spectroscopy.

PubMed

Adams, Kristie M; Marzilli, Patricia A; Marzilli, Luigi G

2007-10-29

Products formed between monoester diphosphates (MDPs) and fac-[Re(CO)3(H2O)3]OTf at pH 3.6 were examined. Such adducts of the fac-[Re(CO)3]+ moiety have an uncommon combination of properties for an "inert" metal center in that sharp NMR signals can be observed, yet the products are equilibrating at rates allowing NMR EXSY cross-peaks to be observed. Thiamine diphosphate (TDP) and uridine 5'-diphosphate (5'-UDP) form 1:1 bidentate {Palpha,Pbeta} chelates, in which the MDP binds Re(I) via Palpha and Pbeta phosphate groups. Asymmetric centers are created at Re(I) (RRe/SRe) and Palpha (Delta/Lambda), leading to four diastereomers. The two mirror pairs of diastereomers (RReDelta/SReLambda) and (RReLambda/SReDelta) for TDP (no ribose) and for all four diastereomers (RReDelta, RReLambda, SReDelta, SReLambda) for 5'-UDP (asymmetric ribose) gave two and four sets of NMR signals for the bound MDP, respectively. 31Palpha-31Palpha EXSY cross-peaks indicate that the fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- isomers interchange slowly on the NMR time scale, with an average k approximately equal to 0.8 s(-1) at 32 degrees C; the EXSY cross-peaks could arise from chirality changes at only Re(I) or at only Palpha. Guanosine 5'-diphosphate (5'-GDP), with a ribose moiety and a Re(I)-binding base, formed both possible diastereomers (RRe and SRe) of the fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- macrochelate, with one slightly more abundant diastereomer suggested to be RRe by Mn2+ ion 1H NMR signal line-broadening combined with distances from molecular models. Interchange of the diastereomers requires that the coordination site of either N7 or Pbeta move to the H2O site. 31Palpha-31Palpha EXSY cross-peaks indicate a k approximately equal to 0.5 s(-1) at 32 degrees C for RRe-to-SRe interchange. The similarity of the rate constants for interchange of fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- and fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- adducts suggest strongly that interchange of Pbeta and H2O coordination positions accounts for the EXSY cross-peaks present in the spectra of all adducts.

Roles of MPBQ-MT in Promoting α/γ-Tocopherol Production and Photosynthesis under High Light in Lettuce

PubMed Central

Tang, Yueli; Fu, Xueqing; Shen, Qian; Tang, Kexuan

2016-01-01

2-methyl-6-phytyl-1, 4-benzoquinol methyltransferase (MPBQ-MT) is a vital enzyme catalyzing a key methylation step in both α/γ-tocopherol and plastoquinone biosynthetic pathway. In this study, the gene encoding MPBQ-MT was isolated from lettuce (Lactuca sativa) by rapid amplification of cDNA ends (RACE), named LsMT. Overexpression of LsMT in lettuce brought about a significant increase of α- and γ-tocopherol contents with a reduction of phylloquinone (vitamin K1) content, suggesting a competition for a common substrate phytyl diphosphate (PDP) between the two biosynthetic pathways. Besides, overexpression of LsMT significantly increased plastoquinone (PQ) level. The increase of tocopherol and plastoquinone levels by LsMT overexpression conduced to the improvement of plants’ tolerance and photosynthesis under high light stress, by directing excessive light energy toward photosynthetic production rather than toward generation of more photooxidative damage. These findings suggest that the role and function of MPBQ-MT can be further explored for enhancing vitamin E value, strengthening photosynthesis and phototolerance under high light in plants. PMID:26867015

Alteration of cell wall polysaccharides through transgenic expression of UDP-Glc 4-epimerase-encoding genes in potato tubers.

PubMed

Huang, Jie-Hong; Kortstee, Anne; Dees, Dianka C T; Trindade, Luisa M; Schols, Henk A; Gruppen, Harry

2016-08-01

Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants.

PubMed Central

Hove-Jensen, B

1996-01-01

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon. PMID:8550505

Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia).

PubMed

Landmann, Christian; Fink, Barbara; Festner, Maria; Dregus, Márta; Engel, Karl-Heinz; Schwab, Wilfried

2007-09-15

The essential oil of lavender (Lavandula angustifolia) is mainly composed of mono- and sesquiterpenes. Using a homology-based PCR strategy, two monoterpene synthases (LaLIMS and LaLINS) and one sesquiterpene synthase (LaBERS) were cloned from lavender leaves and flowers. LaLIMS catalyzed the formation of (R)-(+)-limonene, terpinolene, (1R,5S)-(+)-camphene, (1R,5R)-(+)-alpha-pinene, beta-myrcene and traces of alpha-phellandrene. The proportions of these products changed significantly when Mn(2+) was supplied as the cofactor instead of Mg(2+). The second enzyme LaLINS produced exclusively (R)-(-)-linalool, the main component of lavender essential oil. LaBERS transformed farnesyl diphosphate and represents the first reported trans-alpha-bergamotene synthase. It accepted geranyl diphosphate with higher affinity than farnesyl diphosphate and also produced monoterpenes, albeit at low rates. LaBERS is probably derived from a parental monoterpene synthase by the loss of the plastidial signal peptide and by broadening its substrate acceptance spectrum. The identification and description of the first terpene synthases from L. angustifolia forms the basis for the biotechnological modification of essential oil composition in lavender.

2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase.

PubMed

Baker, C H; Banzon, J; Bollinger, J M; Stubbe, J; Samano, V; Robins, M J; Lippert, B; Jarvi, E; Resvick, R

1991-06-01

It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.

Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric

Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groupsmore » of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.« less

Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

PubMed

Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

2015-11-15

The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phenotypic, antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk.

PubMed

Osman, Kamelia M; Hassan, Hany M; Orabi, Ahmed; Abdelhafez, Ahmed S T

2014-06-01

Studies on the prevalence and virulence genes of Klebsiella mastitis pathogens in a buffalo population are undocumented. Also, the association of rmpA kfu, uge, magA, Aerobactin, K1 and K2 virulent factors with K. pneumoniae buffalo, and cow mastitis is unreported. The virulence of K. pneumoniae was evaluated through both phenotypic and molecular assays. In vivo virulence was assessed by the Vero cell cytotoxicity, suckling mouse assay and mice lethality test. Antimicrobial susceptibility was tested by disk diffusion method. The 45 K. pneumoniae isolates from buffalo (n = 10/232) and cow (n = 35/293) milk were isolated (45/525; 8.6%) and screened via PCR for seven virulence genes encoding uridine diphosphate galactose 4 epimerase encoding gene responsible for capsule and smooth lipopolysaccharide synthesis (uge), siderophores (kfu and aerobactin), protectines or invasins (rmpA and magA), and the capsule and hypermucoviscosity (K1 and K2). The most common virulence genes were rmpA, kfu, uge, and magA (77.8% each). Aerobactin and K1 genes were found at medium rates of 66.7% each and K2 (55.6%). The Vero cell cytotoxicity and LD (50) in mice were found in 100% of isolates. A multidrug resistance pattern was observed for 40% of the antimicrobials. The distribution of virulence profiles indicate a role of rmpA, kfu, uge, magA, Aerobactin, and K1 and K2 in pathogenicity of K. pneumoniae in udder infections and invasiveness, and constitutes a threat for vulnerable animals, even more if they are in combination with antibiotic resistance.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase[OPEN

PubMed Central

Niehaus, Thomas D.; Hasnain, Ghulam; Gidda, Satinder K.; Nguyen, Thuy N.D.; Anderson, Erin M.; Brown, Greg; Yakunin, Alexander F.; de Crécy-Lagard, Valérie; Gregory, Jesse F.

2016-01-01

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms. PMID:27677881

Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria.

PubMed

Choi, Sun Young; Lee, Hyun Jeong; Choi, Jaeyeon; Kim, Jiye; Sim, Sang Jun; Um, Youngsoon; Kim, Yunje; Lee, Taek Soon; Keasling, Jay D; Woo, Han Min

2016-01-01

Metabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value-added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2. Here, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33). This is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular OverMEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.

Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flentke, G.R.; Frey, P.A.

UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent bindingmore » of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.« less

Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.

2012-09-17

Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further ourmore » understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.« less

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

PubMed Central

Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

2016-01-01

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

PubMed

Nguyen, Khiem; Li, Jin; Puthenveetil, Robbins; Lin, Xiaochen; Poe, Michael M; Hsiao, Chia-Hung Christine; Vinogradova, Olga; Wiemer, Andrew J

2017-11-01

Small isoprenoid diphosphates, such as ( E )-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST 296 AA or T 304 A) investigated, confirm that the backbone amide of at least one Thr (Thr 304 ), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr 296/297 ) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM 2 -C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region. © FASEB.

Binding of uridine 5'-diphosphate in the "basic patch" of the zinc deacetylase LpxC and implications for substrate binding.

PubMed

Gennadios, Heather A; Christianson, David W

2006-12-26

LpxC is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of lipid A, a vital component of the outer membrane of Gram-negative bacteria. Accordingly, the inhibition of LpxC is an attractive strategy for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC from Aquifex aeolicus complexed with uridine 5'-diphosphate (UDP), and the 3.1 A resolution structure of LpxC complexed with pyrophosphate. The X-ray crystal structure of the LpxC-UDP complex provides the first view of interactions likely to be exploited by the substrate UDP group in the "basic patch" of the active site. The diphosphate group of UDP makes hydrogen bond interactions with strictly conserved residue K239 as well as solvent molecules. The ribose moiety of UDP interacts with partially conserved residue E197. The UDP uracil group hydrogen bonds with both the backbone NH group and the backbone carbonyl group of E160, and with the backbone NH group of K162 through an intervening water molecule. Finally, the alpha-phosphate and uracil groups of UDP interact with R143 and R262 through intervening water molecules. The structure of LpxC complexed with pyrophosphate reveals generally similar intermolecular interactions in the basic patch. Unexpectedly, diphosphate binding in both complexes is accompanied by coordination to an additional zinc ion, resulting in the identification of a new metal-binding site termed the E-site. The structures of the LpxC-UDP and LpxC-pyrophosphate complexes provide new insights with regard to substrate recognition in the basic patch and metal ion coordination in the active site of LpxC.

Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth.

PubMed

Desai, Janish; Wang, Yang; Wang, Ke; Malwal, Satish R; Oldfield, Eric

2016-10-06

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron-withdrawing aryl-alkyl side chains which inhibited the growth of Gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ∼1-4 μg mL -1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially "rescued" by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (∼2-6 μg mL -1 ) against Gram-positive but not Gram-negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ∼1-2 μg mL -1 . © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Condensation of the isoprenoid and amino precursors in the biosynthesis of domoic acid.

PubMed

Savage, Thomas J; Smith, G Jason; Clark, Amy T; Saucedo, Portia N

2012-01-01

Understanding how environmental signals regulate production of domoic acid in blooms of Pseudo-nitzschia spp. at a molecular level requires description of the biochemical pathway to this kainoid neurotoxin. Precursor feeding studies have suggested domoic acid arises from the condensation of the C(10) isoprenoid geranyl diphosphate with glutamate, but the specific reactions leading to domoic acid from these precursors remain undescribed. Here, we develop a method to derivatize domoic acid with propyl chloroformate that enables gas chromatography-mass spectrometry (GC-MS) analysis to measure incorporation of stable isotopes into domoic acid generated in cultures incubated with isotopically-labeled substrates. We apply this method to demonstrate that both (2)H from [1-(2)H(2)]geraniol are incorporated into domoic acid, suggesting that the condensation of geranyl diphosphate with an amino group occurs by nucleophilic substitution of the diphosphate rather than by oxidation of geraniol to the aldehyde before reaction with an amino group to form an imine. Ultimately, these and similar studies will facilitate the identification of DA biosynthetic enzymes and genes which will enable the study of how environmental factors regulate DA biosynthesis at the molecular level. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mechanism of ribonucleotide reductase from Herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ator, M.A.; Stubbe, J.; Spector, T.

1986-03-15

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of (3'-3H)ADP, (3'-H)UDP, and (5-3H) UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of (3'-3H)ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of (beta-32P)ClUDP with the reductase resulted in themore » production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase.« less

Solanesol Biosynthesis in Plants.

PubMed

Yan, Ning; Liu, Yanhua; Zhang, Hongbo; Du, Yongmei; Liu, Xinmin; Zhang, Zhongfeng

2017-03-23

Solanesol is a non-cyclic terpene alcohol composed of nine isoprene units that mainly accumulates in solanaceous plants. Solanesol plays an important role in the interactions between plants and environmental factors such as pathogen infections and moderate-to-high temperatures. Additionally, it is a key intermediate for the pharmaceutical synthesis of ubiquinone-based drugs such as coenzyme Q10 and vitamin K2, and anti-cancer agent synergizers such as N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl) ethylenediamine (SDB). In plants, solanesol is formed by the 2- C -methyl-d-erythritol 4-phosphate (MEP) pathway within plastids. Solanesol's biosynthetic pathway involves the generation of C5 precursors, followed by the generation of direct precursors, and then the biosynthesis and modification of terpenoids; the first two stages of this pathway are well understood. Based on the current understanding of solanesol biosynthesis, we here review the key enzymes involved, including 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), isopentenyl diphosphate isomerase (IPI), geranyl geranyl diphosphate synthase (GGPPS), and solanesyl diphosphate synthase (SPS), as well as their biological functions. Notably, studies on microbial heterologous expression and overexpression of key enzymatic genes in tobacco solanesol biosynthesis are of significant importance for medical uses of tobacco.

Synthesis, Rietveld refinements, Infrared and Raman spectroscopy studies of the sodium diphosphate NaCryFe1-yP2O7 (0 ≤ y ≤ 1)

NASA Astrophysics Data System (ADS)

Bih, H.; Saadoune, I.; Bih, L.; Mansori, M.; ToufiK, H.; Fuess, H.; Ehrenberg, H.

2016-01-01

In the present study we report on the synthesis and crystal structure studies of NaCryFe1-yP2O7 sodium diphosphate solid solution (0 ≤ y ≤ 1). The X-ray diffraction shows that these compounds are isostructural with NaFeP2O7 and NaCrP2O7 (space group P21/c (C2h5) Z = 4). The Rietveld refinements based on the XRD patterns show the existence of a continuous solid solution over the whole composition range (0 ≤ y ≤ 1). A continuous evolution of the monoclinic unit cell parameters was obtained. The transition metal ions (Cr3+ and/or Fe3+) connect the diphosphate anions forming a three-dimensional network with cages filled by Na+ cations. IR and Raman spectra have been interpreted using factor group analysis. A small shift of the band frequencies is observed when Fe is substituted by Cr. The POP bridge angles are determined from Lazarev's relation and agree well with those deduced from the crystal structure refinement.

Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-D-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-D-Rhamnosyltransferase WbpZ.

PubMed

Wang, Shuo; Hao, Youai; Lam, Joseph S; Vlahakis, Jason Z; Szarek, Walter A; Vinnikova, Anna; Veselovsky, Vladimir V; Brockhausen, Inka

2015-06-15

The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of D-rhamnose (D-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-D-Rha and enzymatically synthesized GDP-D-[(3)H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one D-Rha residue from GDP-D-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring D-mannose (D-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-D-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-rhamnosyltransferase that has significant activity of GDP-D-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a D-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. This is the first characterization of a D-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145.

PubMed

Kato, Hiroki; Tsunematsu, Yuta; Yamamoto, Tsuyoshi; Namiki, Takuya; Kishimoto, Shinji; Noguchi, Hiroshi; Watanabe, Kenji

2016-07-01

To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

Cinnamon and Its Metabolite Sodium Benzoate Attenuate the Activation of p21rac and Protect Memory and Learning in an Animal Model of Alzheimer's Disease.

PubMed

Modi, Khushbu K; Roy, Avik; Brahmachari, Saurabh; Rangasamy, Suresh B; Pahan, Kalipada

2015-01-01

This study underlines the importance of cinnamon, a commonly used natural spice and flavoring material, and its metabolite sodium benzoate (NaB) in attenuating oxidative stress and protecting memory and learning in an animal model of Alzheimer's disease (AD). NaB, but not sodium formate, was found to inhibit LPS-induced production of reactive oxygen species (ROS) in mouse microglial cells. Similarly, NaB also inhibited fibrillar amyloid beta (Aβ)- and 1-methyl-4-phenylpyridinium(+)-induced microglial production of ROS. Although NaB reduced the level of cholesterol in vivo in mice, reversal of the inhibitory effect of NaB on ROS production by mevalonate, and geranylgeranyl pyrophosphate, but not cholesterol, suggests that depletion of intermediates, but not end products, of the mevalonate pathway is involved in the antioxidant effect of NaB. Furthermore, we demonstrate that an inhibitor of p21rac geranylgeranyl protein transferase suppressed the production of ROS and that NaB suppressed the activation of p21rac in microglia. As expected, marked activation of p21rac was observed in the hippocampus of subjects with AD and 5XFAD transgenic (Tg) mouse model of AD. However, oral feeding of cinnamon (Cinnamonum verum) powder and NaB suppressed the activation of p21rac and attenuated oxidative stress in the hippocampus of Tg mice as evident by decreased dihydroethidium (DHE) and nitrotyrosine staining, reduced homocysteine level and increased level of reduced glutathione. This was accompanied by suppression of neuronal apoptosis, inhibition of glial activation, and reduction of Aβ burden in the hippocampus and protection of memory and learning in transgenic mice. Therefore, cinnamon powder may be a promising natural supplement in halting or delaying the progression of AD.

Protein farnesyltransferase isoprenoid substrate discrimination is dependent on isoprene double bonds and branched methyl groups.

PubMed

Micali, E; Chehade, K A; Isaacs, R J; Andres, D A; Spielmann, H P

2001-10-16

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K(m) and analogue hydrophobicity and length, there was no correlation between k(cat) and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially occluding the space occupied by the Ca(1)a(2)X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

PubMed

Edler, Eileen; Stein, Matthias

2017-10-25

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Monochromatic green light induces an aberrant accumulation of geranylgeranyled chlorophylls in plants.

PubMed

Materová, Zuzana; Sobotka, Roman; Zdvihalová, Barbora; Oravec, Michal; Nezval, Jakub; Karlický, Václav; Vrábl, Daniel; Štroch, Michal; Špunda, Vladimír

2017-07-01

Light quality is an important environmental factor affecting the biosynthesis of photosynthetic pigments whose production seems to be affected not only quantitatively but also qualitatively. In this work, we set out to identify unusual pigment detected in leaves of barley (Hordeum vulgare L.) and explain its presence in plants grown under monochromatic green light (GL; 500-590 nm). The chromatographic analysis (HPLC-DAD) revealed that a peak belonging to this unknown pigment is eluted between chlorophyll (Chl) a and b. This pigment exhibited the same absorption spectrum and fluorescence excitation and emission spectra as Chl a. It was negligible in control plants cultivated under white light of the same irradiance (photosynthetic photon flux density of 240 μmol m -2  s -1 ). Mass spectrometry analysis of this pigment (ions m/z = 889 [M-H] - ; m/z = 949 [M+acetic acid-H] - ) indicates that it is Chl a with a tetrahydrogengeranylgeraniol side chain (containing two double bonds in a phytyl side chain; Chl a THGG ), which is an intermediate in Chl a synthesis. In plants grown under GL, the proportion of Chl a THGG to total Chl content rose to approximately 8% and 16% after 7 and 14 days of cultivation, respectively. Surprisingly, plants cultivated under GL exhibited drastically increased concentration of the enzyme geranylgeranyl reductase, which is responsible for the reduction of phytyl chain double bonds in the Chl synthesis pathway. This indicates impaired activity of this enzyme in GL-grown plants. A similar effect of GL on Chl synthesis was observed for distinct higher plant species. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Transcriptomic Analysis of Paulownia Infected by Paulownia Witches'-Broom Phytoplasma

PubMed Central

Zhu, Shui-Fang; Lin, Cai-Li; Tian, Guo-Zhong; Xu, Xia; Zhao, Wen-Jun

2013-01-01

Phytoplasmas are plant pathogenic bacteria that have no cell wall and are responsible for major crop losses throughout the world. Phytoplasma-infected plants show a variety of symptoms and the mechanisms they use to physiologically alter the host plants are of considerable interest, but poorly understood. In this study we undertook a detailed analysis of Paulownia infected by Paulownia witches’-broom (PaWB) Phytoplasma using high-throughput mRNA sequencing (RNA-Seq) and digital gene expression (DGE). RNA-Seq analysis identified 74,831 unigenes, which were subsequently used as reference sequences for DGE analysis of diseased and healthy Paulownia in field grown and tissue cultured plants. Our study revealed that dramatic changes occurred in the gene expression profile of Paulownia after PaWB Phytoplasma infection. Genes encoding key enzymes in cytokinin biosynthesis, such as isopentenyl diphosphate isomerase and isopentenyltransferase, were significantly induced in the infected Paulownia. Genes involved in cell wall biosynthesis and degradation were largely up-regulated and genes related to photosynthesis were down-regulated after PaWB Phytoplasma infection. Our systematic analysis provides comprehensive transcriptomic data about plants infected by Phytoplasma. This information will help further our understanding of the detailed interaction mechanisms between plants and Phytoplasma. PMID:24130859

The sh2-R allele of the maize shrunken-2 locus was caused by a complex chromosomal rearrangement.

PubMed

Kramer, Vance; Shaw, Janine R; Senior, M Lynn; Hannah, L Curtis

2015-03-01

The mutant that originally defined the shrunken - 2 locus of maize is shown here to be the product of a complex chromosomal rearrangement. The maize shrunken-2 gene (sh2) encodes the large subunit of the heterotetrameric enzyme, adenosine diphosphate glucose pyrophosphorylases and a rate-limiting enzyme in starch biosynthesis. The sh2 gene was defined approximately 72 years ago by the isolation of a loss-of-function allele conditioning a shrunken, but viable seed. In subsequent years, the realization that this allele, termed zsh2-R or sh2-Reference, causes an extremely high level of sucrose to accumulate in the developing seed led to a revolution in the sweet corn industry. Now, the vast majority of sweet corns grown throughout the world contain this mutant allele. Through initial Southern analysis followed by genomic sequencing, the work reported here shows that this allele arose through a complex set of events involving at least three breaks of chromosome 3 as well as an intra-chromosomal inversion. These findings provide an explanation for some previously reported, unexpected observations concerning rates of recombination within and between genes in this region.

Low Medium pH Value Enhances Anthocyanin Accumulation in Malus Crabapple Leaves

PubMed Central

Tian, Ji; Jin, Kaina; Yao, Yuncong

2014-01-01

Anthocyanin is a critical factor involved in coloration of plant tissues, but the mechanism how medium pH values affect anthocyanin accumulation in woody plants is unknown. We analyzed anthocyanin composition and the expression of elements encoding anthocyanin and flavonols biosynthesis underlying different medium pH values by using three different leave color type cultivars. HPLC analysis demonstrated that high medium pH values treatment induced a dramatic decrease in the concentration of cyaniding in crabapple leaves. Conversely, the high medium pH values induced up-regulation of the content of flavones and flavonols, suggesting that low pH treatment-induced anthocyanin accumulation. Quantitative real time PCR experiment showed the expression level of anthocyanidin synthase (McANS) and uridine diphosphate glucose flavonoid 3-O-glucosyltransferase (McUFGT) was up-regulated by low pH values treatment, and high medium pH value treatment up-regulate the transcription level of flavonol synthase (McFLS). Meanwhile, several MYB TFs have been suggested in the regulation of pH responses. These results strongly indicate that the low pH treatment-induced anthocyanin accumulation is mediated by the variation of mRNA transcription of the anthocyanin biosynthetic genes. PMID:24914811

Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Lange, Bernd Markus; McCaskill, David G.

2001-01-01

cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (Mentha piperita) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

PubMed

Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

2014-04-11

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.

A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia

PubMed Central

Duriez, Bénédicte; Duquesnoy, Philippe; Escudier, Estelle; Bridoux, Anne-Marie; Escalier, Denise; Rayet, Isabelle; Marcos, Elisabeth; Vojtek, Anne-Marie; Bercher, Jean-François; Amselem, Serge

2007-01-01

Thioredoxins belong to a large family of enzymatic proteins that function as general protein disulfide reductases, therefore participating in several cellular processes via redox-mediated reactions. So far, none of the 18 members of this family has been involved in human pathology. Here we identified TXNDC3, which encodes a thioredoxin–nucleoside diphosphate kinase, as a gene implicated in primary ciliary dyskinesia (PCD), a genetic condition characterized by chronic respiratory tract infections, left–right asymmetry randomization, and male infertility. We show that the disease, which segregates as a recessive trait, results from the unusual combination of the following two transallelic defects: a nonsense mutation and a common intronic variant found in 1% of control chromosomes. This variant affects the ratio of two physiological TXNDC3 transcripts: the full-length isoform and a novel isoform, TXNDC3d7, carrying an in-frame deletion of exon 7. In vivo and in vitro expression data unveiled the physiological importance of TXNDC3d7 (whose expression was reduced in the patient) and the corresponding protein that was shown to bind microtubules. PCD is known to result from defects of the axoneme, an organelle common to respiratory cilia, embryonic nodal cilia, and sperm flagella, containing dynein arms, with, to date, the implication of genes encoding dynein proteins. Our findings, which identify a another class of molecules involved in PCD, disclose the key role of TXNDC3 in ciliary function; they also point to an unusual mechanism underlying a Mendelian disorder, which is an SNP-induced modification of the ratio of two physiological isoforms generated by alternative splicing. PMID:17360648

Enterococcus faecalis phosphomevalonate kinase

PubMed Central

Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

2005-01-01

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni++ affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37°C. The activation energy was ~5.6 kcal/mol. Activity with Mn++, the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). Km values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 μmol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed. PMID:15802646

Bifunctional Homodimeric Triokinase/FMN Cyclase

PubMed Central

Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

2014-01-01

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

Molecular Characterization of Soybean Pterocarpan 2-Dimethylallyltransferase in Glyceollin Biosynthesis: Local Gene and Whole-Genome Duplications of Prenyltransferase Genes Led to the Structural Diversity of Soybean Prenylated Isoflavonoids

PubMed Central

Yoneyama, Keisuke; Akashi, Tomoyoshi; Aoki, Toshio

2016-01-01

Soybean (Glycine max) accumulates several prenylated isoflavonoid phytoalexins, collectively referred to as glyceollins. Glyceollins (I, II, III, IV and V) possess modified pterocarpan skeletons with C5 moieties from dimethylallyl diphosphate, and they are commonly produced from (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(−)-glycinol]. The metabolic fate of (−)-glycinol is determined by the enzymatic introduction of a dimethylallyl group into C-4 or C-2, which is reportedly catalyzed by regiospecific prenyltransferases (PTs). 4-Dimethylallyl (−)-glycinol and 2-dimethylallyl (−)-glycinol are precursors of glyceollin I and other glyceollins, respectively. Although multiple genes encoding (−)-glycinol biosynthetic enzymes have been identified, those involved in the later steps of glyceollin formation mostly remain unidentified, except for (−)-glycinol 4-dimethylallyltransferase (G4DT), which is involved in glyceollin I biosynthesis. In this study, we identified four genes that encode isoflavonoid PTs, including (−)-glycinol 2-dimethylallyltransferase (G2DT), using homology-based in silico screening and biochemical characterization in yeast expression systems. Transcript analyses illustrated that changes in G2DT gene expression were correlated with the induction of glyceollins II, III, IV and V in elicitor-treated soybean cells and leaves, suggesting its involvement in glyceollin biosynthesis. Moreover, the genomic signatures of these PT genes revealed that G4DT and G2DT are paralogs derived from whole-genome duplications of the soybean genome, whereas other PT genes [isoflavone dimethylallyltransferase 1 (IDT1) and IDT2] were derived via local gene duplication on soybean chromosome 11. PMID:27986914

Relative significances of pH and substrate starch level to roles of Streptococcus bovis S1 in rumen acidosis.

PubMed

Chen, Lianmin; Liu, Shimin; Wang, Hongrong; Wang, Mengzhi; Yu, Lihuai

2016-12-01

To clarify the relative importance of pH and substrate starch level in fermentation characteristics and regulatory mechanism of Streptococcus bovis S1 in rumen acidosis, an in vitro fermentation of three levels of soluble starch (1, 3 and 9 g/L) was established with pH in the media were maintained constant at 5.5 or 6.5. The results showed that the dominant product of S. bovis S1 was lactate at both pH, the production depended on the starch level, and more lactate was produced at pH 6.5 than that at pH 5.5 (P < 0.001). At pH 5.5, the activity of lactate dehydrogenase (LDH) and α-amylase (α-AMY), their abundances, the relative expressions of LDH, PFL (gene encoding pyruvate formate-lyase), CCPA (gene encoding global catabolite control protein A) and α-AMY genes were higher than those at pH 6.5 (P < 0.05), whereas the concentration of fructose-1,6-diphosphate (FDP) was lower. The activity of LDH, α-AMY and FDP, and the relative expressions of LDH, PFL, CCPA and α-AMY genes were, in general, positively related to the starch level. The canonical regression analysis indicated that the pH had more profound effect compared with the starch level, in terms of the acid productions, enzyme activity and gene expressions. It was concluded that the fermentation of S. bovis was regulated at the transcription level in response to both pH and substrate starch concentration, but more sensitive to pH changes.

Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

PubMed

Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

2013-06-01

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

Crystal structure of Rb{sub 2}Mn{sub 3}(H{sub 2}O){sub 2}[P{sub 2}O{sub 7}]{sub 2}, a new representative of the family of hydrated diphosphates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiriukhina, G. V., E-mail: g-biralo@yandex.ru; Yakubovich, O. V.; Dimitrova, O. V.

2016-09-15

The crystal structure of Rb{sub 2}Mn{sub 3}(H{sub 2}O){sub 2}[P{sub 2}O{sub 7}]{sub 2}, a new phase obtained in the form of single crystals under hydrothermal conditions in the MnCl{sub 2}–Rb{sub 3}PO{sub 4}–H{sub 2}O system, is determined by X-ray diffraction (Xcalibur-S-CCD diffractometer, R = 0.0270): a = 9.374(2), b = 8.367(2), c = 9.437(2) Å, ß = 99.12(2)°, space group P2{sub 1}/c, Z = 2, D{sub x} = 3.27 g/cm{sup 3}. A correlation between the unit-cell parameters and the size of cations forming the crystal structures of isostructural A{sub 2}M{sub 3}(H{sub 2}O){sub 2}[P{sub 2}O{sub 7}]{sub 2} diphosphates (A = K, NH{sub 4},more » Rb, or Na; {sub M} = Mn, Fe, Co, or Ni) is revealed. It is shown that, due to the topological similarity, the structures of diphosphates and orthophosphates of the farringtonite structural type can undergo mutual transformations.« less

Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway*

PubMed Central

Onono, Fredrick; Subramanian, Thangaiah; Sunkara, Manjula; Subramanian, Karunai Leela; Spielmann, H. Peter; Morris, Andrew J.

2013-01-01

Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition. PMID:23908355

Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

PubMed

Karner, Susanne; Shi, Shaojun; Fischer, Christine; Schaeffeler, Elke; Neurath, Markus F; Herrlinger, Klaus R; Hofmann, Ute; Schwab, Matthias

2010-04-01

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Association of Higher Plasma Vitamin D Binding Protein and Lower Free Calcitriol Levels with Tenofovir Disoproxil Fumarate Use and Plasma and Intracellular Tenofovir Pharmacokinetics: Cause of a Functional Vitamin D Deficiency?

PubMed Central

Kiser, Jennifer J.; Stephensen, Charles B.; Hazra, Rohan; Flynn, Patricia M.; Wilson, Craig M.; Rutledge, Brandy; Bethel, James; Pan, Cynthia G.; Woodhouse, Leslie R.; Van Loan, Marta D.; Liu, Nancy; Lujan-Zilbermann, Jorge; Baker, Alyne; Kapogiannis, Bill G.; Gordon, Catherine M.

2013-01-01

Tenofovir disoproxil fumarate (TDF) causes bone, endocrine, and renal changes by an unknown mechanism(s). Data are limited on tenofovir pharmacokinetics and these effects. Using baseline data from a multicenter study of HIV-infected youth on stable treatment with regimens containing TDF (n = 118) or lacking TDF (n = 85), we measured cross-sectional associations of TDF use with markers of renal function, vitamin D-calcium-parathyroid hormone balance, phosphate metabolism (tubular reabsorption of phosphate and fibroblast growth factor 23 [FGF23]), and bone turnover. Pharmacokinetic-pharmacodynamic associations with plasma tenofovir and intracellular tenofovir diphosphate concentrations were explored among those receiving TDF. The mean age was 20.9 (standard deviation [SD], 2.0) years; 63% were male; and 52% were African American. Compared to the no-TDF group, the TDF group showed lower mean estimated glomerular filtration rates and tubular reabsorption of phosphate, as well as higher parathyroid hormone and 1,25-dihydroxy vitamin D [1,25-OH(2)D] levels. The highest quintile of plasma tenofovir concentrations was associated with higher vitamin D binding protein, lower free 1,25-OH(2)D, higher 25-OH vitamin D, and higher serum calcium. The highest quintile of intracellular tenofovir diphosphate concentration was associated with lower FGF23. Higher plasma tenofovir concentrations were associated with higher vitamin D binding protein and lower free 1,25-OH(2)D, suggesting a functional vitamin D deficiency explaining TDF-associated increased parathyroid hormone. The finding of lower FGF23 accompanying higher intracellular tenofovir diphosphate suggests that different mechanisms mediate TDF-associated changes in phosphate handling. Separate pharmacokinetic properties may be associated with distinct TDF toxicities: tenofovir with parathyroid hormone and altered calcium balance and tenofovir diphosphate with hypophosphatemia and FGF23 regulation. (The clinical trial registration number for this study is NCT00490412 and is available online at http://clinicaltrials.gov/ct2/show/NCT00490412.) PMID:24002093

Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus†

PubMed Central

Thibodeaux, Christopher J.; Mansoorabadi, Steven O.; Kittleman, William; Chang, Wei-chen; Liu, Hung-wen

2011-01-01

The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steady-state and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect (D2OVmax = 1.5) was measured on vo in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity (DVmax = 1.8) and on the decay rate of the flavin intermediate (Dks = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2H]-IPP. Taken together, these data suggest that the C2–H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures. PMID:18229948

Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

PubMed

Hsieh, Wei-Yu; Sung, Tzu-Ying; Wang, Hsin-Tzu; Hsieh, Ming-Hsiun

2014-09-01

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes. © 2014 American Society of Plant Biologists. All Rights Reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Modolo, Luzia V.; Li, Lenong; Pan, Haiyun

The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 {angstrom} resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides amore » basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.« less

Hair cell specific NTPDase6 immunolocalisation in vestibular end organs: potential role of purinergic signaling in vestibular sensory transduction.

PubMed

O'Keeffe, Mary G; Thorne, Peter R; Housley, Gary D; Robson, Simon C; Vlajkovic, Srdjan M

2012-01-01

A complex extracellular nucleotide signalling system acting on P2 receptors is involved in regulation of cochlear function in the mammalian inner ear. Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are ectonucleotidases that regulate P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from the CD39/ENTPD family (NTPDase1-8) are expressed in the adult rat cochlea, but their expression and distribution in the vestibular end organ is unknown. This report demonstrates selective expression of NTPDase6 by rat vestibular hair cells. Hair cells transducing both angular acceleration (crista ampullaris) and static head position (maculae of the utricle and saccule) exhibited strong immunolabelling with a bias towards the sensory pole and in particular, the hair cell bundle. NTPDase6 is an intracellular enzyme that can be released in a soluble form from cell cultures and shows an enzymatic preference for nucleoside 5'-diphosphates, such as guanosine 5'-diphosphate (GDP) and uridine 5'-diphosphate (UDP). The main function of NTPDase6 may be the regulation of nucleotide levels in cellular organelles by regulating the conversion of nucleotides to nucleosides. NTPDase6 immunolocalisation in the vestibular end organ could be linked to the regulation of P2 receptor signalling and sensory transduction, including maintenance of vestibular hair bundles.

Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli.

PubMed

Liu, Wei; Zhang, Rubing; Tian, Ning; Xu, Xin; Cao, Yujing; Xian, Mo; Liu, Huizhou

2015-01-01

Geraniol is a valuable acyclic monoterpene alcohol and has many applications in the perfume industries, pharmacy and others. It has been hypothesized that phosphatases can convert geranyl diphosphate (GPP) into geraniol. However, whether and which phosphatases can transform GPP to geraniol has remained unanswered up till now. In this paper, the catalysis abilities of 4 different types of phosphatases were studied with GPP as substrate in vitro. They are bifunctional diacylglycerol diphosphate phosphatase (DPP1) and lipid phosphate phosphatase (LPP1) from Saccharomyces cerevisiae, ADP-ribose pyrophosphatase (NudF) and alkaline phosphatase (PhoA) from Escherichia coli. The results show that just PhoA from E. coli can convert GPP into geraniol. Moreover, in order to confirm the ability of PhoA in vivo, the heterologous mevalonate pathway and geranyl diphosphate synthase gene from Abies grandis were co-overexpressed in E. coli with PhoA gene and 5.3 ± 0.2 mg/l geraniol was produced from glucose in flask-culture. Finally, we also evaluated the fed-batch fermentation of this engineered E. coli and a maximum concentration of 99.3 mg/l geraniol was produced while the conversion efficiency of glucose to geranoid (gram to gram) was 0.51%. Our results offer a new option for geraniol biosynthesis and promote the industrial bio-production of geraniol.

Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli

PubMed Central

Liu, Wei; Zhang, Rubing; Tian, Ning; Xu, Xin; Cao, Yujing; Xian, Mo; Liu, Huizhou

2015-01-01

Geraniol is a valuable acyclic monoterpene alcohol and has many applications in the perfume industries, pharmacy and others. It has been hypothesized that phosphatases can convert geranyl diphosphate (GPP) into geraniol. However, whether and which phosphatases can transform GPP to geraniol has remained unanswered up till now. In this paper, the catalysis abilities of 4 different types of phosphatases were studied with GPP as substrate in vitro. They are bifunctional diacylglycerol diphosphate phosphatase (DPP1) and lipid phosphate phosphatase (LPP1) from Saccharomyces cerevisiae, ADP-ribose pyrophosphatase (NudF) and alkaline phosphatase (PhoA) from Escherichia coli. The results show that just PhoA from E. coli can convert GPP into geraniol. Moreover, in order to confirm the ability of PhoA in vivo, the heterologous mevalonate pathway and geranyl diphosphate synthase gene from Abies grandis were co-overexpressed in E. coli with PhoA gene and 5.3 ± 0.2 mg/l geraniol was produced from glucose in flask-culture. Finally, we also evaluated the fed-batch fermentation of this engineered E. coli and a maximum concentration of 99.3 mg/l geraniol was produced while the conversion efficiency of glucose to geranoid (gram to gram) was 0.51%. Our results offer a new option for geraniol biosynthesis and promote the industrial bio-production of geraniol. PMID:26091008

Surface acidic amino acid of Pseudomonas/Halomonas chimeric nucleoside diphosphate kinase leads effective recovery from heat-denaturation.

PubMed

Tokunaga, Hiroko; Arakawa, Tsutomu; Tokunaga, Masao

2013-07-01

One of the hallmarks of halophilic properties is reversibility of thermal unfolding. A nucleoside diphosphate kinase (NDK) from a moderate halophile Halomonas sp. 593 (HaNDK) follows this behavior. His-tagged chimeric NDK (HisPaHaNDK) consisting of an N-terminal half of a non-halophilic Pseuodomonas aeruginosa NDK (PaNDK) and a Cterminal half of HaNDK loses this reversible property, indicating a critical role of the N-terminal portion of PaNDK in determining the reversibility of the chimeric protein. Various mutations were introduced at Arg45 and Lys61, based on the model NDK structure. It appears that having Glu at position 45 is critical in conferring the thermal reversibility to HisPa- HaNDK chimeric protein.

Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum

PubMed Central

2010-01-01

Background Little genomic or trancriptomic information on Ganoderma lucidum (Lingzhi) is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST) library. Methods A cDNA library was constructed from the G. lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs) was preformed by online analysis. Results A total of 1,023 clones were randomly selected from the G. lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE) and farnesyl-diphosphate synthase (FPS) were identified in this EST collection. Several candidate genes, such as hydrophobin, MOB2, profilin and PHO84 were detected for the first time in G. lucidum. Thirteen (13) potential SSR-motif microsatellite loci were also identified. Conclusion The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum. PMID:20230644

Discovery and Characterization of a Group of Fungal Polycyclic Polyketide Prenyltransferases

PubMed Central

Chooi, Yit-Heng; Wang, Peng; Fang, Jinxu; Li, Yanran; Wu, Katherine; Wang, Pin; Tang, Yi

2014-01-01

The prenyltransferase (PTase) gene vrtC was proposed to be involved in viridicatumtoxin (1) biosynthesis in Penicillium aethiopicum. Targeted gene deletion and reconstitution of recombinant VrtC activity in vitro established that VrtC is a geranyl transferase that catalyzes a regiospecific Friedel-Crafts alkylation of the naphthacenedione carboxamide intermediate 2 at carbon 6 with geranyl diphosphate (GPP). VrtC can function in the absence of divalent ions and can utilize similar naphthacenedione substrates, such as the acetyl-primed TAN-1612 (4). Genome mining using the VrtC protein sequence leads to the identification of a homologous group of PTase genes in the genomes of human and animal-associated fungi. Three enzymes encoded by this new subgroup of PTase genes from Neosartorya fischeri, Microsporum canis and Trichophyton tonsurans were shown to be able to catalyze transfer of dimethylallyl to several tetracyclic naphthacenedione substrates in vitro. In total, seven C5- or C10-prenylated naphthacenedione compounds were generated. The regioselectivity of these new polycyclic PTases (pcPTases) was confirmed by characterization of product 9 obtained from biotransformation of 4 in Escherichia coli expressing the N. fischeri pcPTase gene. The discovery of this new subgroup of PTases extends our enzymatic tools for modifying polycyclic compounds and enables genome mining of new prenylated polyketides. PMID:22590971

Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

PubMed

Li, Huan-Jun; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2016-01-10

Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetics of platelet inhibitor treatment

PubMed Central

Trenk, Dietmar; Hochholzer, Willibald

2014-01-01

Dual antiplatelet therapy with aspirin and a P2Y12 receptor antagonist is the standard of care in patients undergoing percutaneous coronary intervention (PCI) and in patients with acute coronary syndromes (ACS) because this regimen has markedly decreased the rate of cardiovascular events. The substantial variability in pharmacodynamic response as well as the moderate antiplatelet efficacy of clopidogrel has raised major concerns, since high on-clopidogrel platelet reactivity has consistently been associated with increased risk for ischaemic events in PCI patients. Baseline demographic and clinical variables contributing to the observed variability have been identified. Besides this, research within the past decade has focused on the impact of genetic polymorphisms encoding transport systems or enzymes involved in the absorption and metabolism of these drugs. Loss-of-function polymorphisms in CYP2C19 are the strongest individual variables affecting pharmacokinetics and antiplatelet response to clopidogrel, but explain no more than 5 to 12% of the variability in adenosine diphosphate-induced platelet aggregation on clopidogrel. No genetic variables contributing to clinical outcomes of patients treated with the newer P2Y12 receptor antagonists, prasugrel or ticagrelor, have been identified so far. This review aims to provide an update on the current status of genotype-based personalized therapy with clopidogrel. PMID:23981082

Associations of MDR1, TBXA2R, PLA2G7, and PEAR1 genetic polymorphisms with the platelet activity in Chinese ischemic stroke patients receiving aspirin therapy.

PubMed

Peng, Ling-Ling; Zhao, Yuan-Qi; Zhou, Zi-Yi; Jin, Jing; Zhao, Min; Chen, Xin-Meng; Chen, Ling-Yan; Cai, Ye-Feng; Li, Jia-Li; Huang, Min

2016-11-01

Aspirin resistance has an incidence of 5%-65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A 2 receptor (TXA 2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A 2 (Lp-PLA 2 , encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB 2 ELISA kit. Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233-0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080-6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than noncarriers (odds ratio=8.233, 95% CI: 1.590-42.638). A considerable portion (11.66%) of Chinese ischemic stroke patients are insensitive to aspirin treatment, which may be correlated with the MDR1 C3435T, TBXA2R (rs1131882), and PLA2G7 (rs1051931-rs7756935) polymorphisms.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

PubMed

Chauhan, Indira Singh; Kaur, Jaspreet; Krishna, Shagun; Ghosh, Arpita; Singh, Prashant; Siddiqi, Mohammad Imran; Singh, Neeloo

2015-11-21

Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.

Lovastatin inhibits proliferation of anaplastic thyroid cancer cells through up-regulation of p27 by interfering with the Rho/ROCK-mediated pathway.

PubMed

Zhong, Wen-Bin; Hsu, Sung-Po; Ho, Pei-Yin; Liang, Yu-Chih; Chang, Tien-Chun; Lee, Wen-Sen

2011-12-01

Previously, we demonstrated that lovastatin, a HMG-CoA reductase inhibitor, induced apoptosis, differentiation, and inhibition of invasiveness of human anaplastic thyroid carcinoma cells (ATCs). Here, we further examined the effect of lovastatin on the growth of ARO cells. Lovastatin (0-20μM) concentration-dependently decreased cell number in cultured ATC and arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that lovastatin caused an increase of the protein level of p27 and cyclin-dependent kinase (CDK)4 and a decrease of the protein level of cyclin A2, cyclin D3, and phosphorylated Rb (pRb), but did not significantly change the protein levels of p21, cyclins D1 and E, and CDK2, in ARO cells. The formation of the CDK2-p27 complex was increased and the CDK2 activity was decreased in the lovastatin-treated ARO cells. Pretreatment of ARO cells with a p27, but not p21, antisense oligonucleotide prevented the lovastatin-induced G0/G1 arrest in ARO cells. The lovastatin-induced growth inhibition and translocation of RhoA and Rac1 in ARO cells were completely prevented by mevalonate and partially by geranylgeranyl pyrophosphate. Treatment of ARO cells with Y27632, an inhibitor of Rho-associated kinase, abolished the GGPP-mediated prevention of lovastatin-induced anti-proliferation and up-regulation and prolonged degradation of p27. Taken together, these data suggest that lovastatin treatment caused a reduction of Rho geranylgeranylation, which in turn increased the expression and stability of p27, and then inhibited ARO cell proliferation. These data suggest that lovastatin merits further investigation as multipotent therapy for treatment ATC. Copyright © 2011 Elsevier Inc. All rights reserved.

The Best Enzyme Investigation Ever? Probably.

ERIC Educational Resources Information Center

Cooper, Phil

2000-01-01

Uses alkaline phosphate to remove the phosphate group from phenolphthalein diphosphate. Discusses problems which include the interference of ambient light and temperature variation. Provides detailed information about the apparatus and the experimental procedure. (ASK)

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

PubMed

Morandim, Andreia de A; Bergamo, Débora Cristina B; Kato, Massuo Jorge; Cavalheiro, Alberto José; Bolzani, Vanderlan da S; Furlan, Maysa

2005-01-01

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.

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.).

PubMed

Yuan, Kejun; Wang, Changjun; Xin, Li; Zhang, Anning; Ai, Chengxiang

2013-07-25

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar "White Winter Pearmain". When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4°C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples. Copyright © 2013 Elsevier B.V. All rights reserved.

Enzymatic synthesis of polymers containing nicotinamide mononucleotide

NASA Technical Reports Server (NTRS)

Liu, Rihe

1995-01-01

Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

Enzymatic Synthesis of Polymers Containing Nicotinamide Mononucleotide

NASA Technical Reports Server (NTRS)

Liu, Rihe; Orgel, Leslie E.

1995-01-01

Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase.

PubMed Central

Keasling, J D; Bertsch, L; Kornberg, A

1993-01-01

An exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] activity has recently been purified to homogeneity from a mutant strain of Escherichia coli which lacks the principal exopoly(P)ase. The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five tryptic digestion fragments of the purified protein are found in the translated gppA gene, (ii) the size of the protein (100 kDa) agrees with published values for GPP, and (iii) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure. The enzyme liberates orthophosphate by processive hydrolysis of the phosphoanyhydride bonds of polyphosphate [poly(P)] chains (1000 residues) or by hydrolysis of the 5'-gamma-phosphate of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) to guanosine 5'-diphosphate 3'-diphosphate (ppGpp or "magic spot"). The Km for long-chain poly(P) as a substrate (approximately 0.5 nM) is far lower than that for pppGpp (0.13 mM); the kcat for the poly(P)ase activity is 1.1 s-1 and that for pppGpp hydrolase is 0.023 s-1. These and other findings direct attention to possible functions of poly(P) in the response of E. coli to stresses and deprivations. Images Fig. 4 PMID:8394006

Interception of the Enzymatic Conversion of Farnesyl Diphosphate to 5-Epi-Aristolochene by Using a Fluoro Substrate Analogue: 1-Fluorogermacrene A from (2E,6Z)-6-Fluorofarnesyl Diphosphate**

PubMed Central

Faraldos, Juan A.; Zhao, Yuxin; O'Maille, Paul E.; Noel, Joseph P.; Coates, Robert M.

2009-01-01

Tobacco 5-epi-aristolochene synthase (TEAS) catalyzes the MgII-dependent cyclizations and rearrangements of (E,E)-farnesyl diphosphate (PP) to the bicyclic sesquiterpene hydrocarbon via a tightly bound (+)-germacrene A as a deprotonated intermediate. With the native enzyme, only a few percent of the putative germacrene A intermediate is released from the active site during the catalytic cycle. 6-Fluorofarnesyl PP was designed and synthesized with the aim of arresting the cyclization-rearrangement mechanism en route to 5-epi-aristolochene. Indeed, incubation of (2E,6Z)-6-fluorofarnesyl PP with recombinant TEAS afforded (-)-1-fluororogermacrene A as the sole product in 58% yield. Steady-state kinetic experiments with farnesyl PP and the 6-fluoro analogue showed that the overall catalytic efficiencies (kcat/Km) are essentially the same for both substrates. 1-Fluorogermacrene A was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR and 1H NMR data, and by heat-induced Cope rearrangement to (+)-1-fluoro-β-elemene. 1H NMR spectra at room temperature revealed that this (E,E)-configured fluorocyclodecadiene exists in solution as a 7:3 mixture of UU and UD conformers. 1-Fluorogermacrene A underwent trifluoroacetic acid-catalyzed cyclization to give three 1α-fluoroselinene isomers at a rate estimated to be about 1000 times slower than that of the similar cyclization of (+)-germacrene A to the parent selinenes. PMID:17886322

Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth, respiration and carbon metabolism.

PubMed

Dorion, Sonia; Clendenning, Audrey; Rivoal, Jean

2017-03-01

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40-fold difference in NDPK activity. Root growth, O 2 uptake, flux of carbon between sucrose and CO 2 , levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP-glucose and cellulose contents. The activation state of ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP-glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Chloroquine diphosphate bearing dextran nanoparticles augmented drug delivery and overwhelmed drug resistance in Plasmodium falciparum parasites.

PubMed

Kashyap, Aman; Kaur, Rupinder; Baldi, Ashish; Jain, Upendra Kumar; Chandra, Ramesh; Madan, Jitender

2018-07-15

Chloroquine diphosphate (CHQ) is primarily used for the treatment of Plasmodium falciparum malaria at the dose of 500mg orally or 10mg/kg parenterally. However, point mutations in Plasmodiumfalciparum chloroquine resistance transporter (PfCRT) protein and Plasmodium falciparum multidrug resistance protein 1 (Pfmdr1) localized in digestive vacuole membrane, are responsible for CHQ resistance. Therefore, in present investigation, dextran nanoparticles bearing chloroquine diphosphate (CHQ-DEX-NPs) were formulated by solvent diffusion method of size below 70nm with zeta-potential of -20.1±3.2mV. FT-IR, DSC and PXRD techniques confirmed the successful loading of drug in nanomatrix system with amorphous attributes. In vitro drug release analysis indicated the Higuchi pattern with diffusion controlled drug release. The IC 50 of CHQ-DEX-NPs in sensitive (3D7) and resistant (RKL9) Plasmodium falciparum strains was estimated to be 0.031-μg/ml and 0.13-μg/ml significantly lower than 0.059-μg/ml and 0.36-μg/ml of CHQ. The augmented therapeutic efficacy of CHQ-DEX-NPs may be credited to deposition of tailored nanoparticles in food vacuoles of malaria parasites owing to the affinity of parasite towards DEX that consequently lower the drug resistance and improved the therapeutic index. In conclusion, CHQ-DEX-NPs must be evaluated under a set of stringent in vivo parameters to establish its therapeutic efficacy in preclinical model. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates.

PubMed

Eriksson, Andreas C; Whiss, Per A

2009-04-01

Platelet adhesion is a complex and important event for prevention of blood loss after vessel injury. This study investigated fundamental adhesive mechanisms occurring in an in-vitro assay developed for the measurement of static adhesion of human platelets in plasma. The aim was to gain methodological knowledge that could be used for interpretations of results from other studies using this specific assay. Involvement of adhesive receptors was investigated by the use of various antibodies as well as therapeutic drugs (abciximab, eptifibatide and tirofiban). Inhibitors of adenosine 5'-diphosphate receptors (cangrelor, MRS2179) and of thromboxane A(2) signalling (BM-531) were used to estimate the role of autocrine activation. Adhesion to collagen was found to be mainly mediated by alpha(2)beta(1) and to some extent by alpha(IIb)beta(3). Adhesion to fibrinogen was mediated by alpha(IIb)beta(3). In addition, adenosine 5'-diphosphate-induced adhesion to albumin was dependent on alpha(IIb)beta(3). Furthermore, experiments with cangrelor and BM-531 showed that the majority of the adhesive interactions tested were dependent on adenosine 5'-diphosphate or thromboxane A(2). We conclude that the mechanisms of adhesion measured by the static platelet adhesion assay are in accordance with the current knowledge regarding platelet activation and adhesion. Despite its simplicity, we suggest that this adhesion assay could be used as a screening device for the study of the influence of various surfaces and soluble substances on platelet adhesion.

Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit

2008-07-28

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg{sup 2+} as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used tomore » probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 {angstrom} (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.« less

Probing the active center of benzaldehyde lyase with substitutions and the pseudo-substrate analog benzoylphosphonic acid methyl ester

PubMed Central

Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

2009-01-01

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these type of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analog of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (PDB ID: 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase. PMID:18570438

The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

PubMed Central

Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

2015-01-01

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

Effect of uridine derivatives on myocardial stunning during postischemic reperfusion of rat heart.

PubMed

Sapronov, N S; Eliseev, V V; Rodionova, O M

2000-10-01

Uridine and uridine-5'-monophosphate prevent myocardial stunning during postischemic reperfusion of isolated rat heart. Uridine-5'-diphosphate does not prevent postischemic myocardial dysfunction, while uridine-5'-triphosphate aggravates it.

Terpenoids and Their Biosynthesis in Cyanobacteria

PubMed Central

Pattanaik, Bagmi; Lindberg, Pia

2015-01-01

Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids. PMID:25615610

Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation.

PubMed

Zeidler, J; Lichtenthaler, H K

2001-06-01

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.

Cytosolic ppGpp accumulation induces retarded plant growth and development.

PubMed

Ihara, Yuta; Masuda, Shinji

2016-01-01

In bacteria a second messenger, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), synthesized upon nutrient starvation, controls many gene expressions and enzyme activities, which is necessary for growth under changeable environments. Recent studies have shown that ppGpp synthase and hydrolase are also conserved in eukaryotes, although their functions are not well understood. We recently showed that ppGpp-overaccumulation in Arabidopsis chloroplasts results in robust growth under nutrient-limited conditions, demonstrating that the bacterial-like stringent response at least functions in plastids. To test if ppGpp also functions in the cytosol, we constructed the transgenic Arabidopsis expressing Bacillus subtilis ppGpp synthase gene yjbM. Upon induction of the gene, the mutant synthesizes ∼10-20-fold higher levels of ppGpp, and its fresh weight was reduced to ˜80% that of the wild type. These results indicate that cytosolic ppGpp negatively regulates plant growth and development.

Bone scanning in severe external otitis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levin, W.J.; Shary, J.H. 3d.; Nichols, L.T.

1986-11-01

Technetium99 Methylene Diphosphate bone scanning has been considered an early valuable tool to diagnose necrotizing progressive malignant external otitis. However, to our knowledge, no formal studies have actually compared bone scans of otherwise young, healthy patients with severe external otitis to scans of patients with clinical presentation of malignant external otitis. Twelve patients with only severe external otitis were studied with Technetium99 Diphosphate and were compared to known cases of malignant otitis. All scans were evaluated by two neuroradiologists with no prior knowledge of the clinical status of the patients. Nine of the 12 patients had positive bone scans withmore » many scans resembling those reported with malignant external otitis. Interestingly, there was no consistent correlation between the severity of clinical presentation and the amount of Technetium uptake. These findings suggest that a positive bone scan alone should not be interpreted as indicative of malignant external otitis.« less

Na7Cr4(P2O7)4PO4

PubMed Central

Bourguiba Fakhar, Noura; Zid, Mohamed Faouzi; Driss, Ahmed

2013-01-01

The title compound, hepta­sodium tetra­chromium(III) tetra­kis­(diphosphate) orthophosphate, was synthesized by solid-state reaction. Its structure is isotypic with that of Na7 M 4(P2O7)4PO4 (M = In, Al) compounds and is made up from a three-dimensional [(CrP2O7)4PO4]7− framework with channels running along [001]. The three Na+ cations are located in the voids of the framework. One of the cations is situated on a general position, one is equally disordered around a twofold rotation axis and one is on a fourfold rotoinversion axis. The isolated PO4 tetra­hedron of the anionic framework is also situated on the -4 axis. Structural relationships between the title compound and different diphosphates containing MP2O11 units (M = Mo, V) are discussed. PMID:23723751

Two types of alcohol dehydrogenase from Perilla can form citral and perillaldehyde.

PubMed

Sato-Masumoto, Naoko; Ito, Michiho

2014-08-01

Studies on the biosynthesis of oil compounds in Perilla will help in understanding regulatory systems of secondary metabolites and in elucidating reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, an aldo-keto reductase (AKR) and a geraniol dehydrogenase (GeDH), which are thought to participate in the biosynthesis of perilla essential oil components, such as citral and perillaldehyde, were isolated from three pure lines of perilla. These enzymes shared high amino acid sequence identity within the genus Perilla, and were expressed regardless of oil type. The overall reaction from geranyl diphosphate to citral was performed in vitro using geraniol synthase and GeDH to form a large proportion of citral and relatively little geraniol as reaction products. The biosynthetic pathway from geranyl diphosphate to citral, the main compound of citral-type perilla essential oil, was established in this study. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamics of Monoterpene Formation in Spike Lavender Plants.

PubMed

Mendoza-Poudereux, Isabel; Kutzner, Erika; Huber, Claudia; Segura, Juan; Arrillaga, Isabel; Eisenreich, Wolfgang

2017-12-19

The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender ( Lavandula latifolia Med) on the basis of 13 CO₂-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13 CO₂ in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the MEP pathway. Surprisingly, overexpression of HMGR did not have significant impact upon the crosstalk between the MVA and MEP pathways indicating that the MEP route is the preferred pathway for the synthesis of C5 monoterpene precursors in spike lavender.

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

PubMed

Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

1995-07-01

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Electron density reactivity indexes of the tautomeric/ionization forms of thiamin diphosphate.

PubMed

Jaña, Gonzalo A; Delgado, Eduardo J

2013-09-01

The generation of the highly reactive ylide in thiamin diphosphate catalysis is analyzed in terms of the nucleophilicity of key atoms, by means of density functional calculations at X3LYP/6-31++G(d,p) level of theory. The Fukui functions of all tautomeric/ionization forms are calculated in order to assess their reactivity. The results allow to conclude that the highly conserved glutamic residue does not protonate the N1' atom of the pyrimidyl ring, but it participates in a strong hydrogen bonding, stabilizing the eventual negative charge on the nitrogen, in all forms involved in the ylide generation. This condition provides the necessary reactivity on key atoms, N4' and C2, to carry out the formation of the ylide required to initiate the catalytic cycle of ThDP-dependent enzymes. This study represents a new approach for the ylide formation in ThDP catalysis.

Off-plane polarization ordering in metal chalcogen diphosphates from bulk to monolayer

NASA Astrophysics Data System (ADS)

Song, Wenshen; Fei, Ruixiang; Yang, Li

2017-12-01

Vertically (off-plane) ferroelectric ordering in ultrathin films has been pursued for decades. We predict the existence of intrinsic vertical polarization orderings in ultrathin metal chalcogen-diphosphates (MCDs). Taking CuInP2Se6 as an example, the first-principles calculation and electrostatic-energy model show that, under the open-circuit boundary condition, the ground state of bulk CuInP2Se6 is ferroelectric (FE) while that of monolayer is antiferroelectric (AFE), and the critical thickness for this FE/AFE transition is around six layers. Interestingly, under the closed-circuit boundary condition, the FE state can hold even for monolayer. Particularly, because of the small energy difference but the large barrier between FE and AFE orderings, the FE state can be stabilized in a free-standing monolayer, giving rise to intrinsic, off-plane two-dimensional ferroelectrics. Applying Monte Carlo simulations, we further calculate the ferroelectric Curie temperature (Tc) and electric hysteresis.

Quenching of graphene quantum dots fluorescence by alkaline phosphatase activity in the presence of hydroquinone diphosphate.

PubMed

Pereira da Silva Neves, Marta Maria; González-García, María Begoña; Pérez-Junquera, Alejandro; Hernández-Santos, David; Fanjul-Bolado, Pablo

2018-05-01

In this work, a turn-off photoluminescent sensing proof-of-concept based on blue luminescent graphene quantum dots (GQDs) as the fluorescent probe was developed. For that purpose, GQDs optical response was related with the catalytic enzymatic activity of alkaline phosphatase (ALP), in the presence of hydroquinone diphosphate (HQDP). The hydrolysis of HQDP by ALP generated hydroquinone (HQ). The oxidation of HQ, enzymatically produced, to p-benzoquinone (BQ) resulted in the quenching of GQDs fluorescence (FL). Therefore, the developed luminescent sensing mechanism allowed the FL quenching with ALP activity to be related and thus quantified the concentration of ALP down to 0.5 nM of enzyme. This innovative design principle appears as a promising tool for the development of enzymatic sensors based on ALP labeling with fluorescent detection or even for direct ALP luminescent quantification in an easy, fast and sensitive manner. Copyright © 2018 John Wiley & Sons, Ltd.

Synthesis and Characterization of a New Hydroquinone Derivative: Disodium p-Phenylene Diisostearyl Diphosphate.

PubMed

Hisama, Masayoshi; Matsuda, Sanae; Arai, Junichi; Masui, Katsunobu; Yamamura, Haruo

2015-01-01

A novel amphiphilic hydroquinone derivative having a C18 alkyl chain phosphate attached to the hydroquinone (HQ) moiety was chemically synthesized. The thermal stability, distribution between organic and aqueous phases, and in vitro skin permeability were evaluated. This HQ derivative was identified as disodium p-phenylene diisostearyl diphosphate (HQ-2P2IS) by UV, infrared, mass, and nuclear magnetic resonance spectroscopies. Product HQ-2P2IS was obtained in good yield (56%), and it exhibited satisfactory stability in neutral solution, comparable to that of HQ. Its skin permeability was also higher than that of HQ. HQ-2P2IS is susceptible to enzymatic hydrolysis by tissue phosphatase, which releases HQ in the skin tissues. Thus, these characteristics indicate that the novel hydroquinone derivative presented herein, i.e., HQ-2P2IS, may serve as an effective pro-hydroquinone for skin care applications.

A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

PubMed Central

Takami, Tomonori; Fang, Yue; Zhou, Xin; Jaiseng, Wurentuya; Ma, Yan; Kuno, Takayoshi

2012-01-01

Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level. PMID:23145048

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8.

PubMed

Bengoechea, José Antonio; Pinta, Elise; Salminen, Tiina; Oertelt, Clemens; Holst, Otto; Radziejewska-Lebrecht, Joanna; Piotrowska-Seget, Zofia; Venho, Reija; Skurnik, Mikael

2002-08-01

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.

A Novel GDP-d-glucose Phosphorylase Involved in Quality Control of the Nucleoside Diphosphate Sugar Pool in Caenorhabditis elegans and Mammals*

PubMed Central

Adler, Lital N.; Gomez, Tara A.; Clarke, Steven G.; Linster, Carole L.

2011-01-01

The plant VTC2 gene encodes GDP-l-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-d-glucose to GDP and d-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-d-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-d-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-d-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-d-glucose in the C10F3.4 mutant worms, suggesting that the GDP-d-glucose phosphorylase may function to remove GDP-d-glucose formed by GDP-d-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological d-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals. PMID:21507950

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

PubMed

Adler, Lital N; Gomez, Tara A; Clarke, Steven G; Linster, Carole L

2011-06-17

The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

Gene Sets for Utilization of Primary and Secondary Nutrition Supplies in the Distal Gut of Endangered Iberian Lynx

PubMed Central

Alcaide, María; Messina, Enzo; Richter, Michael; Bargiela, Rafael; Peplies, Jörg; Huws, Sharon A.; Newbold, Charles J.; Golyshin, Peter N.; Simón, Miguel A.; López, Guillermo; Yakimov, Michail M.; Ferrer, Manuel

2012-01-01

Recent studies have indicated the existence of an extensive trans-genomic trans-mural co-metabolism between gut microbes and animal hosts that is diet-, host phylogeny- and provenance-influenced. Here, we analyzed the biodiversity at the level of small subunit rRNA gene sequence and the metabolic composition of 18 Mbp of consensus metagenome sequences and activity characteristics of bacterial intra-cellular extracts, in wild Iberian lynx (Lynx pardinus) fecal samples. Bacterial signatures (14.43% of all of the Firmicutes reads and 6.36% of total reads) related to the uncultured anaerobic commensals Anaeroplasma spp., which are typically found in ovine and bovine rumen, were first identified. The lynx gut was further characterized by an over-representation of ‘presumptive’ aquaporin aqpZ genes and genes encoding ‘active’ lysosomal-like digestive enzymes that are possibly needed to acquire glycerol, sugars and amino acids from glycoproteins, glyco(amino)lipids, glyco(amino)glycans and nucleoside diphosphate sugars. Lynx gut was highly enriched (28% of the total glycosidases) in genes encoding α-amylase and related enzymes, although it exhibited low rate of enzymatic activity indicative of starch degradation. The preponderance of β-xylosidase activity in protein extracts further suggests lynx gut microbes being most active for the metabolism of β-xylose containing plant N-glycans, although β-xylosidases sequences constituted only 1.5% of total glycosidases. These collective and unique bacterial, genetic and enzymatic activity signatures suggest that the wild lynx gut microbiota not only harbors gene sets underpinning sugar uptake from primary animal tissues (with the monotypic dietary profile of the wild lynx consisting of 80–100% wild rabbits) but also for the hydrolysis of prey-derived plant biomass. Although, the present investigation corresponds to a single sample and some of the statements should be considered qualitative, the data most likely suggests a tighter, more coordinated and complex evolutionary and nutritional ecology scenario of carnivore gut microbial communities than has been previously assumed. PMID:23251564

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway.

PubMed

Hsiao, Yu-Yun; Tsai, Wen-Chieh; Kuoh, Chang-Sheng; Huang, Tian-Hsiang; Wang, Hei-Chia; Wu, Tian-Shung; Leu, Yann-Lii; Chen, Wen-Huei; Chen, Hong-Hwa

2006-07-13

Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis. The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. This systems biology program combined chemical analysis, genomics and bioinformatics to elucidate the scent biosynthesis pathway and identify the relevant genes. It integrates the forward and reverse genetic approaches to knowledge discovery by which researchers can study non-model plants.

Pheno- and Genotyping of Hopanoid Production in Acidobacteria

PubMed Central

Damsté, Jaap S. Sinninghe; Rijpstra, W. Irene C.; Dedysh, Svetlana N.; Foesel, Bärbel U.; Villanueva, Laura

2017-01-01

Hopanoids are pentacyclic triterpenoid lipids synthesized by different bacterial groups. Methylated hopanoids were believed to be exclusively synthesized by cyanobacteria and aerobic methanotrophs until the genes encoding for the methylation at the C-2 and C-3 position (hpnP and hpnR) were found to be widespread in the bacterial domain, invalidating their use as specific biomarkers. These genes have been detected in the genome of the Acidobacterium “Ca. Koribacter versatilis,” but our knowledge of the synthesis of hopanoids and the presence of genes of their biosynthetic pathway in other member of the Acidobacteria is limited. We analyzed 38 different strains of seven Acidobacteria subdivisions (SDs 1, 3, 4, 6, 8, 10, and 23) for the presence of C30 hopenes and C30+ bacteriohopane polyols (BHPs) using the Rohmer reaction. BHPs and/or C30 hopenes were detected in all strains of SD1 and SD3 but not in SD4 (excepting Chloracidobacterium thermophilum), 6, 8, 10, and 23. This is in good agreement with the presence of genes required for hopanoid biosynthesis in the 31 available whole genomes of cultivated Acidobacteria. All genomes encode the enzymes involved in the non-mevalonate pathway ultimately leading to farnesyl diphosphate but only SD1 and 3 Acidobacteria and C. thermophilum encode all three enzymes required for the synthesis of squalene, its cyclization (shc), and addition and modification of the extended side chain (hpnG, hpnH, hpnI, hpnJ, hpnO). In almost all strains, only tetrafunctionalized BHPs were detected; three strains contained variable relative abundances (up to 45%) of pentafunctionalized BHPs. Only “Ca. K. versatilis” contained methylated hopanoids (i.e., 2,3-dimethyl bishomohopanol), although in low (<10%) amounts. These genes are not present in any other Acidobacterium, consistent with the absence of methylated BHPs in the other examined strains. These data are in agreement with the scattered occurrence of methylated BHPs in other bacterial phyla such as the Alpha-, Beta-, and Gammaproteobacteria and the Cyanobacteria, limiting their biomarker potential. Metagenomes of Acidobacteria were also examined for the presence of genes required for hopanoid biosynthesis. The complete pathway for BHP biosynthesis was evident in SD2 Acidobacteria and a group phylogenetically related to SD1 and SD3, in line with the limited occurrence of BHPs in acidobacterial cultures. PMID:28642737

Study of the Crystal Structures of Sodium Magnesium and Sodium Nickel Diphosphates

NASA Astrophysics Data System (ADS)

Erragh, Fatima; Boukhari, Ali; Abraham, Francis; Elouadi, Brahim

2000-07-01

Single-crystal X-ray crystallography studies have shown that diphosphates Na3.64Mg2.18(P2O7)2 and Na3.64Ni2.18(P2O7)2 crystallize with the same structural type and the same space group Poverline1. Their triclinic lattice parameters are equal to a=10.901 (2), b=9.765 (2), c=6.382 (1) Å, α=112.43 (1)°, β=99.64 (1)°, γ=107.53 (1)°, Z=2 and a=10.889 (5), b=9.705 (4), c=6.358 (4) Å, α=112.46 (4)°, β=99.92 (4)°, γ=107.54 (4)°, Z=2, respectively. The structure could be regarded as a packing of diphosphate groups [P2O7]4- and [MO6] octahedra (M=Mg, Ni) delimiting cavities and tunnels which host sodium cations. The tunnels are running along [001]. The structure is characterized by mixed (Na, M) sites with an occupation factor ratio equal to ca. 0.82/0.18. Sodium cations are located in five different sites: two cavities (one penta- and the other octa-coordinated) totally occupied and three octahedral interstices, which are partially filled by Na(3), Na(4), and Na(5) according to the respective occupation factors of 0.15, 0.42, and 0.25 for Na3.64Mg2.18(P2O7)2 and 0.13, 0.40, and 0.29 in the case of Na3.64Ni2.18(P2O7)2.

A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2'-azido-2'-deoxynucleoside diphosphates.

PubMed

Sjöberg, B M; Gräslund, A; Eckstein, F

1983-07-10

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a tyrosine radical which is essential for enzyme activity. In the reaction between ribonucleotide reductase and the substrate analogue 2'-azido-2'-deoxycytidine 5'-diphosphate a new transient radical is formed. The EPR characteristics of this new radical species are consistent with a localization of the unpaired electron at the sugar moiety of the nucleotide. The radical shows hyperfine couplings to a hydrogen and a nitrogen nucleus, the latter probably being part of the azide substituent. The formation of the nucleotide radical in this suicidal reaction is concomitant with the decay of the tyrosine radical of the B2 subunit. Kinetic data argue for a first (pseudosecond) order decay of the B2 radical via generation of the nucleotide radical followed by a slower first order decay of the nucleotide radical. End products in the reaction are cytosine and radical-free protein B2. In the reaction between bacteriophage T4 ribonucleotide reductase and 2'-azido-2'-deoxycytidine 5'-diphosphate an identical nucleotide radical is formed. The present results are consistent with the hypothesis that the appearance and structure of the transient radical mimic stages in the normal reaction pathway of ribonucleotide reductase, postulated to proceed via 3'-hydrogen abstraction and cation radical formation of the substrate nucleotide (Stubbe, J., and Ackles, D. (1980) J. Biol. Chem. 255, 8027-8030). The nucleotide radical described here might be equivalent to such a cation radical intermediate.

CTP synthase 1, a smooth muscle-sensitive therapeutic target for effective vascular repair

PubMed Central

Tang, Rui; Cui, Xiao-Bing; Wang, Jia-Ning; Chen, Shi-You

2013-01-01

Objective Vascular remodeling due to smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, anti-neointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, due to their non-specific effect on endothelial cells (EC). The objective of this study was to identify a therapeutic target that differentially regulates SMC and EC proliferation. Approach and Results By using both rat balloon-injury and mouse wire-injury models, we identified CTP synthase (CTPS) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization due to induction of CTP synthesis salvage pathway enzymes nucleoside diphosphate kinase A and B in ECs. Diphosphate kinase B appeared to preserve EC proliferation via utilization of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelization in vivo. Conclusions Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair following injury. PMID:24008161

Enzymatic regeneration of adenosine triphosphate cofactor

NASA Technical Reports Server (NTRS)

Marshall, D. L.

1974-01-01

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

Synthesis, crystal structures and optical properties of two congruent-melting isotypic diphosphates: LiM{sub 3}P{sub 2}O{sub 7} (M=Na, K)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi Yunjing; Wang Ying; Graduate University of the Chinese Academy of Sciences, Beijing 100039

2013-01-15

Two new isotypic diphosphates, LiNa{sub 3}P{sub 2}O{sub 7} (1) and LiK{sub 3}P{sub 2}O{sub 7} (2), have been synthesized by conventional solid-state reaction. The single-crystal X-ray structural analyses have shown that they crystallize in the orthorhombic space group C222{sub 1} (No. 20) with the unit cells: a=5.4966(2) A, b=9.1365(4) A, c=12.2764(5) A for compound 1 and a=6.0373(14) A, b=9.339(2) A, c=13.292(3) A for compound 2. The LiM{sub 3}P{sub 2}O{sub 7} (M=Na, K) consist of two-dimensional [LiP{sub 2}O{sub 7}]{sup 3-} layers, which are composed by LiO{sub 4} tetrahedral and diphosphate groups, the Na or K atoms are filled in the interlayers andmore » balance the charge. Second harmonic generation (SHG) on powder samples have been measured using Kurtz and Perry techniques. Thermal analyses, IR spectroscopy, UV-vis-NIR diffuse reflectance spectra, and band structure calculations are performed on the reported compounds. - Graphical Abstract: LiM{sub 3}P{sub 2}O{sub 7} (M=Na, K) consists of a two-dimensional infinite [LiP{sub 2}O{sub 7}]{sup 3-} layer, which is composed by LiO{sub 4} tetrahedra and diphosphate groups. Highlights: Black-Right-Pointing-Pointer LiNa{sub 3}P{sub 2}O{sub 7} and LiK{sub 3}P{sub 2}O{sub 7} are new compounds in the Li{sub 2}O-M{sub 2}O (M=Na, K)-P{sub 2}O{sub 5} systems. Black-Right-Pointing-Pointer Crystal structures of LiNa{sub 3}P{sub 2}O{sub 7} and LiK{sub 3}P{sub 2}O{sub 7} consist of two-dimensional [LiP{sub 2}O{sub 7}]{sup 3-} layers. Black-Right-Pointing-Pointer LiNa{sub 3}P{sub 2}O{sub 7} and LiK{sub 3}P{sub 2}O{sub 7} are congruent melting compounds.« less

Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.

PubMed

Jiang, Guo-Zhen; Yao, Ming-Dong; Wang, Ying; Zhou, Liang; Song, Tian-Qing; Liu, Hong; Xiao, Wen-Hai; Yuan, Ying-Jin

2017-05-01

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20 WW (Erg20 F96W-N127W ), co-expression of the reverse fusion of Erg20 ww /t3CrGES and another copy of Erg20 WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20 WW and the free Erg20 WW . Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering. Copyright © 2017. Published by Elsevier Inc.

Electron attachment to DNA single strands: gas phase and aqueous solution.

PubMed

Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

2007-01-01

The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers such as glycosidic bond breakage. However, the radical anions of the pyrimidine nucleotides with high VDE are expected to be electronically stable. Thus the base-centered radical anions of the pyrimidine nucleotides might be the possible intermediates for DNA single-strand breakage.

Generation of blue chrysanthemums by anthocyanin B-ring hydroxylation and glucosylation and its coloration mechanism.

PubMed

Noda, Naonobu; Yoshioka, Satoshi; Kishimoto, Sanae; Nakayama, Masayoshi; Douzono, Mitsuru; Tanaka, Yoshikazu; Aida, Ryutaro

2017-07-01

Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)-glucose:anthocyanin 3',5'- O -glucosyltransferase gene, in addition to the expression of the Canterbury bells F3'5'H . Newly synthesized 3',5'-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed "copigmentation," with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.

Acetone utilization by sulfate-reducing bacteria: draft genome sequence of Desulfococcus biacutus and a proteomic survey of acetone-inducible proteins.

PubMed

Gutiérrez Acosta, Olga B; Schleheck, David; Schink, Bernhard

2014-07-11

The sulfate-reducing bacterium Desulfococcus biacutus is able to utilize acetone for growth by an inducible degradation pathway that involves a novel activation reaction for acetone with CO as a co-substrate. The mechanism, enzyme(s) and gene(s) involved in this acetone activation reaction are of great interest because they represent a novel and yet undefined type of activation reaction under strictly anoxic conditions. In this study, a draft genome sequence of D. biacutus was established. Sequencing, assembly and annotation resulted in 159 contigs with 5,242,029 base pairs and 4773 predicted genes; 4708 were predicted protein-encoding genes, and 3520 of these had a functional prediction. Proteins and genes were identified that are specifically induced during growth with acetone. A thiamine diphosphate-requiring enzyme appeared to be highly induced during growth with acetone and is probably involved in the activation reaction. Moreover, a coenzyme B12- dependent enzyme and proteins that are involved in redox reactions were also induced during growth with acetone. We present for the first time the genome of a sulfate reducer that is able to grow with acetone. The genome information of this organism represents an important tool for the elucidation of a novel reaction mechanism that is employed by a sulfate reducer in acetone activation.

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids.

PubMed

Harada, Hisashi; Maoka, Takashi; Osawa, Ayako; Hattan, Jun-Ichiro; Kanamoto, Hirosuke; Shindo, Kazutoshi; Otomatsu, Toshihiko; Misawa, Norihiko

2014-04-01

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3'-hydroxylase (CrtZ) and β,β-carotenoid 4,4'-ketolase (4,4'-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8%) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2% of total carotenoids), astaxanthin monoester (18.2%), and the free forms of astaxanthin (10.0%) and the other ketocarotenoids (17.5%), which indicated that artificial ketocarotenoids corresponded to 94.9% of total carotenoids (230 μg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8%) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.

Characterization of Streptococcus pneumoniae thymidylate kinase: steady-state kinetics of the forward reaction and isothermal titration calorimetry.

PubMed Central

Petit, Chantal M; Koretke, Kristin K

2002-01-01

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis. The tmk gene from the bacterial pathogen Streptococcus pneumoniae was identified. The gene, encoding a 212-amino-acid polypeptide (23352 Da), was cloned and overexpressed in Escherichia coli with an N-terminal hexahistidine tag. The enzyme was purified to homogeneity, and characterized in the forward reaction. The pH profile of TMK indicates that its activity is optimal at pH 8.5. The substrate specificity of the enzyme was examined; it was found that not only ATP, but also dATP and to a lesser extent CTP, could act as phosphate donors, and dTMP and dUMP could serve as phosphate acceptors. Furthermore, AZT-MP (3'-azido-3'-deoxythymidine 5'-monophosphate) was shown not to be a substrate for S. pneumoniae TMK. Steady-state kinetics and inhibition studies with adenosine 5'-[beta-thio]diphosphate and dTDP in addition to isothermal titration calorimetry were performed. The data showed that binding follows an ordered pathway, in which ATP binds first with a K(m) of 235 +/- 46 microM and a K(d) of 116 +/- 3 microM, and dTMP binds secondly with a K(m) of 66 +/- 12 microM and a K(d) of 53 +/- 2 microM. PMID:11964185

Suppression of the E. coli SOS response by dNTP pool changes.

PubMed

Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J; Schaaper, Roel M

2015-04-30

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Megakaryocyte and platelet abnormalities in a patient with a W33C mutation in the conserved SH3-like domain of myosin heavy chain IIA.

PubMed

Kahr, Walter H A; Savoia, Anna; Pluthero, Fred G; Li, Ling; Christensen, Hilary; De Rocco, Daniela; Traivaree, Chanchai; Butchart, Sheila E; Curtin, Julie; Stollar, Elliott J; Forman-Kay, Julie D; Blanchette, Victor S

2009-12-01

Heterozygous mutations in MYH9, which encodes non-muscle myosin heavy chain IIA (MHC-IIA), result in autosomal dominant inherited MYH9-related disorders characterised by macro-thrombocytopenia, granulocyte inclusions, variable sensorineural deafness, cataracts and nephritis. MHC-IIA is assembled into a complex consisting of two pairs of light chains and two heavy chains, where the latter contain a neck region, SH3-like, motor and rod domains. We describe a patient with a Trp33Cys missense mutation in the SH3-like domain of MHC-IIA. Abnormal platelet function was observed using platelet aggregometry with the agonists epinephrine and adenosine diphosphate (ADP). Patient granulocytes and megakaryocytes, but not platelets, contained abnormal MHC-IIA inclusions visualised by confocal immunofluorescence or electron microscopy. Megakaryocytes grown in culture were smaller and contained hypolobulated nuclei compared to controls. Bone marrow-derived megakaryocytes revealed a preponderance of immature forms, the presence of structurally diverse inclusion bodies, and frequent emperipolesis as assessed by electron microscopy. Platelets and leukocytes contained indistinguishable amounts of total MHC-IIA determined by immunoblotting. Molecular modelling studies indicated that mutation of Trp33 destabilises the interface between the SH3-like and motor domain of MHC-IIA, which is close to previously described motor domain mutations, implying an important structural and/or functional role for this region in MHC-IIA.

Production of trichodiene by Trichoderma harzianum alters the perception of this biocontrol strain by plants and antagonized fungi.

PubMed

Malmierca, Mónica G; McCormick, Susan P; Cardoza, Rosa E; Alexander, Nancy J; Monte, Enrique; Gutiérrez, Santiago

2015-08-01

Trichothecenes are phytotoxic sesquiterpenic mycotoxins that can act as virulence factors in plant diseases. Harzianum A (HA) is a non-phytotoxic trichothecene produced by Trichoderma arundinaceum. The first step in HA biosynthesis is the conversion of farnesyl diphosphate to trichodiene (TD), a volatile organic compound (VOC), catalysed by a sesquiterpene synthase encoded by the tri5 gene. Expression of tri5 in the biocontrol strain Trichoderma harzianum CECT 2413 resulted in production of TD in parallel with a reduction of ergosterol biosynthesis and an unexpected increase in the level of squalene. Transformants expressing tri5 displayed low chitinase activity and induced expression of Botrytis cinerea BOT genes, although their total antagonistic potential against phytopathogenic fungi was not reduced. VOCs released by the tri5-transformant induced expression of tomato defence genes related to salicylic acid (SA), and TD itself strongly induced the expression of SA-responsive genes and reduced the development of lateral roots. Together, these results suggest that TD acts as a signalling VOC in the interactions of Trichoderma with plants and other microorganisms by modulating the perception of this fungus to a given environment. Moreover, the TD ability to induce systemic defences indicates that complex trichothecene structures may not be necessary for inducing such responses. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Molecular structure and differential function of choline kinases CHKα and CHKβ in musculoskeletal system and cancer.

PubMed

Chen, Xi; Qiu, Heng; Wang, Chao; Yuan, Yu; Tickner, Jennifer; Xu, Jiake; Zou, Jun

2017-02-01

Choline, a hydrophilic cation, has versatile physiological roles throughout the body, including cholinergic neurotransmission, memory consolidation and membrane biosynthesis and metabolism. Choline kinases possess enzyme activity that catalyses the conversion of choline to phosphocholine, which is further converted to cytidine diphosphate-coline (CDP-choline) in the biosynthesis of phosphatidylcholine (PC). PC is a major constituent of the phospholipid bilayer which constitutes the eukaryotic cell membrane, and regulates cell signal transduction. Choline Kinase consists of three isoforms, CHKα1, CHKα2 and CHKβ, encoded by two separate genes (CHKA(Human)/Chka(Mouse) and CHKB(Human)/Chkb(Mouse)). Both isoforms have similar structures and enzyme activity, but display some distinct molecular structural domains and differential tissue expression patterns. Whilst Choline Kinase was discovered in early 1950, its pivotal role in the development of muscular dystrophy, bone deformities, and cancer has only recently been identified. CHKα has been proposed as a cancer biomarker and its inhibition as an anti-cancer therapy. In contrast, restoration of CHKβ deficiency through CDP-choline supplements like citicoline may be beneficial for the treatment of muscular dystrophy, bone metabolic diseases, and cognitive conditions. The molecular structure and expression pattern of Choline Kinase, the differential roles of Choline Kinase isoforms and their potential as novel therapeutic targets for muscular dystrophy, bone deformities, cognitive conditions and cancer are discussed. Copyright © 2016. Published by Elsevier Ltd.

RED BLOOD CELL PRESERVATION.

DTIC Science & Technology

A number of erythrocyte phosphate metabolites (adenosine tri- and diphosphates, guanosine triphosphate, 2,3- diphosphoglycerate ) affected heme-heme...maintained at higher concentrations than those stored in CPD blood; DPG levels were greater in CPD blood. These differences were due to the pH of the

Problem-Solving Test: Catalytic Activities of a Human Nuclear Enzyme

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5'-end and 3'-end, bacteriophage,…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneousmore » leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.« less

Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

PubMed

Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

2016-05-01

Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067.

PubMed

Figueroa, Dominique B; Tillotson, Joseph; Li, Maoji; Piwowar-Manning, Estelle; Hendrix, Craig W; Holtz, Timothy H; Bokoch, Kevin; Bekker, Linda-Gail; van Griensven, Frits; Mannheimer, Sharon; Hughes, James P; Grant, Robert M; Bumpus, Namandjé N

2018-01-01

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif.

PubMed

Hsiao, Yu-Yun; Jeng, Mei-Fen; Tsai, Wen-Chieh; Chuang, Yu-Chen; Li, Chia-Ying; Wu, Tian-Shung; Kuoh, Chang-Sheng; Chen, Wen-Huei; Chen, Hong-Hwa

2008-09-01

Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg(2+) and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.

Analysis of polyphosphates in fish and shrimps tissues by two different ion chromatography methods: implications on false-negative and -positive findings.

PubMed

Kaufmann, A; Maden, K; Leisser, W; Matera, M; Gude, T

2005-11-01

Inorganic polyphosphates (di-, tri- and higher polyphosphates) can be used to treat fish, fish fillets and shrimps in order to improve their water-binding capacity. The practical relevance of this treatment is a significant gain of weight caused by the retention/uptake of water and natural juice into the fish tissues. This practice is legal; however, the use of phosphates has to be declared. The routine control testing of fish for the presence of polyphosphates, produced some results that were difficult to explain. One of the two analytical methods used determined low diphosphate concentrations in a number of untreated samples, while the other ion chromatography (IC) method did not detect them. This initiated a number of investigations: results showed that polyphosphates in fish and shrimps tissue undergo a rapid enzymatic degradation, producing the ubiquitous orthophosphate. This led to the conclusion that sensitive analytical methods are required in order to detect previous polyphosphate treatment of a sample. The polyphosphate concentrations detected by one of the analytical methods could not be explained by the degradation of endogenous high-energy nucleotides like ATP into diphosphate, but by a coeluting compound. Further investigations by LC-MS-MS proved that the substance responsible for the observed peak was inosine monophsosphate (IMP) and not as thought the inorganic diphosphate. The method producing the false-positive result was modified and both methods were ultimately able to detect polyphosphates well separated from natural nucleotides. Polyphosphates could no longer be detected (<0.5 mg kg-1) after modification of the analytical methodology. The relevance of these findings lies in the fact that similar analytical methods are employed in various control laboratories, which might lead to false interpretation of measurements.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

PubMed

Kumar, Ramasamy P; Morehouse, Benjamin R; Matos, Jason O; Malik, Karan; Lin, Hongkun; Krauss, Isaac J; Oprian, Daniel D

2017-03-28

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with K I values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The K I values are similar to the K M for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (k cat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s -1 , respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Sustainable heterologous production of terpene hydrocarbons in cyanobacteria.

PubMed

Formighieri, Cinzia; Melis, Anastasios

2016-12-01

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial application. However, the slow catalytic activity of terpene synthases (k cat  = 4 s -1 or slower) makes them noncompetitive for the pool of available substrate, thereby limiting the rate and yield of product generation. Work in this paper applied transformation technologies in Synechocystis for the heterologous production of β-phellandrene (monoterpene) hydrocarbons. Conditions were defined whereby expression of the β-phellandrene synthase (PHLS), as a CpcB·PHLS fusion protein with the β-subunit of phycocyanin, accounted for up to 20 % of total cellular protein. Moreover, CpcB·PHLS was heterologously co-expressed with enzymes of the mevalonic acid (MVA) pathway and geranyl-diphosphate synthase, increasing carbon flux toward the terpenoid biosynthetic pathway and enhancing substrate availability. These improvements enabled yields of 10 mg of β-phellandrene per g of dry cell weight generated in the course of a 48-h incubation period, or the equivalent of 1 % β-phellandrene:biomass (w:w) carbon-partitioning ratio. The work helped to identify prerequisites for the efficient heterologous production of terpene hydrocarbons in cyanobacteria: (i) requirement for overexpression of the heterologous terpene synthase, so as to compensate for the slow catalytic turnover of the enzyme, and (ii) enhanced endogenous carbon partitioning toward the terpenoid biosynthetic pathway, e.g., upon heterologous co-expression of the MVA pathway, thereby supplementing the native metabolic flux toward the universal isopentenyl-diphosphate and dimethylallyl-diphosphate terpenoid precursors. The two prerequisites are shown to be critical determinants of yield in the photosynthetic CO 2 to terpene hydrocarbons conversion process.

Abacavir has no prothrombotic effect on platelets in vitro.

PubMed

Diallo, Yacouba L; Ollivier, Véronique; Joly, Véronique; Faille, Dorothée; Catalano, Giovanna; Jandrot-Perrus, Martine; Rauch, Antoine; Yeni, Patrick; Ajzenberg, Nadine

2016-12-01

HIV patients exposed to abacavir have an increased risk of myocardial infarction, with contradictory results in the literature. The aim of our study was to determine whether abacavir has a direct effect on platelet activation and aggregation using platelets from healthy donors and from HIV-infected patients under therapy with an undetectable viral load. Platelet-rich plasma (PRP) or whole blood from healthy donors was treated with abacavir (5 or 10 μg/mL) or its active metabolite carbovir diphosphate. Experiments were also performed using blood of HIV-infected patients (n = 10) with an undetectable viral load. Platelet aggregation was performed on PRP by turbidimetry and under high shear conditions at 4000 s -1 . Platelet procoagulant potential was analysed by measuring thrombin generation by thrombinography. Abacavir and carbovir diphosphate significantly increased the aggregation of platelets from healthy donors induced by collagen at 2 μg/mL (P = 0.002), but not at 0.5 μg/mL. No effect of abacavir or carbovir diphosphate was observed on platelet aggregation induced by other physiological agonists or by high shear stress, or on thrombin generation. Pretreatment of blood from HIV-infected patients with abacavir produced similar results. Our results suggest that abacavir does not significantly influence platelet activation in vitro when incubated with platelets from healthy donors or from HIV-infected patients. It is, however, not excluded that a synergistic effect with other drugs could promote platelet activation and thereby play a role in the pathogenesis of myocardial infarction. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

PubMed

Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

2004-03-09

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

PubMed

Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

2017-05-01

Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221. Copyright © 2017. Published by Elsevier Ltd.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding.

PubMed

Souza, Tatiana A C B; Trindade, Daniel M; Tonoli, Celisa C C; Santos, Camila R; Ward, Richard J; Arni, Raghuvir K; Oliveira, Arthur H C; Murakami, Mário T

2011-07-01

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Effects of sulfate aerosols upon cardiopulmonary function in squirrel monkeys (final report). (Second year final report: effects of nitrate and /or sulfate aerosols upon cardiopulmonary function)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenberg, H.L.; Avol, E.L.; Bailey, R.M.

1977-06-24

Squirrel monkeys were exposed to nominal concentrations of 2.5 mg/cu m of the following generated aerosols: zinc ammonium sulfate at both low (40%) and high (85%) nominal relative humidities, histamine diphosphate at low relative humidity, and ammonium bisulfate at low relative humidity. There were few statistically significant changes in oscillatory resistance, however, several trends toward increased resistance were present. Additional studies were performed using two different aerosol nebulizers to produce histamine diphosphate particles in two distinct size distributions, an order of magnitude different in size. These results, supported by data collected during sulfur dioxide exposures of squirrel monkeys, are usedmore » to discuss the suitability of Saimiri sciureus as a sensitive indicator species and oscillatory resistance as a valuable measurement of proximal airway changes. Development of the esophageal balloon technique as a means of measuring compliance and resistance in the unanesthetized squirrel monkey is discussed.« less

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli: Production of Jet Fuel Precursor Monoterpenoids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendez-Perez, Daniel; Alonso-Gutierrez, Jorge; Hu, Qijun

Monoterpenes (C 10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C 15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of Escherichia coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP productionmore » but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes.« less

A homogeneous nucleic acid hybridization assay based on strand displacement.

PubMed Central

Vary, C P

1987-01-01

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Images PMID:3309890

Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

PubMed

Yamaguchi, Nobuo; Yoshinaga, Masafumi; Kamino, Kei; Ueki, Tatsuya

2016-06-01

Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

Adenylate Energy Charge in Escherichia coli During Growth and Starvation

PubMed Central

Chapman, Astrid G.; Fall, Lana; Atkinson, Daniel E.

1971-01-01

The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types. PMID:4333317

p53 regulates the mevalonate pathway in human glioblastoma multiforme

PubMed Central

Laezza, C; D'Alessandro, A; Di Croce, L; Picardi, P; Ciaglia, E; Pisanti, S; Malfitano, A M; Comegna, M; Faraonio, R; Gazzerro, P; Bifulco, M

2015-01-01

The mevalonate (MVA) pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, we provided evidence that p53 induces the expression of a group of enzymes of the MVA pathway including 3′-hydroxy-3′-methylglutaryl-coenzyme A reductase, MVA kinase, farnesyl diphosphate synthase and farnesyl diphosphate farnesyl transferase 1, in the human glioblastoma multiforme cell line, U343 cells, and in normal human astrocytes, NHAs. Genetic and pharmacologic perturbation of p53 directly influences the expression of these genes. Furthermore, p53 is recruited to the gene promoters in designated p53-responsive elements, thereby increasing their transcription. Such effect was abolished by site-directed mutagenesis in the p53-responsive element of promoter of the genes. These findings highlight another aspect of p53 functions unrelated to tumor suppression and suggest p53 as a novel regulator of the MVA pathway providing insight into the role of this pathway in cancer progression. PMID:26469958

Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

NASA Astrophysics Data System (ADS)

Both, P.; Green, A. P.; Gray, C. J.; Šardzík, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.; Richardson, D.; Field, R. A.; Widmalm, G.; Flitsch, S. L.; Eyers, C. E.

2014-01-01

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents.

PubMed

Masini, Tiziana; Hirsch, Anna K H

2014-12-11

Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli: Production of Jet Fuel Precursor Monoterpenoids

DOE PAGES

Mendez-Perez, Daniel; Alonso-Gutierrez, Jorge; Hu, Qijun; ...

2017-05-18

Monoterpenes (C 10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C 15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of Escherichia coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP productionmore » but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes.« less

Dynamics of Monoterpene Formation in Spike Lavender Plants

PubMed Central

Kutzner, Erika; Huber, Claudia; Segura, Juan; Arrillaga, Isabel

2017-01-01

The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender (Lavandula latifolia Med) on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13CO2 in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the MEP pathway. Surprisingly, overexpression of HMGR did not have significant impact upon the crosstalk between the MVA and MEP pathways indicating that the MEP route is the preferred pathway for the synthesis of C5 monoterpene precursors in spike lavender. PMID:29257083

Optimization of thermophilic trans-isoprenyl diphosphate synthase expression in Escherichia coli by response surface methodology.

PubMed

Piccolomini, Angelica A; Fiabon, Alex; Borrotti, Matteo; De Lucrezia, Davide

2017-01-01

We optimized the heterologous expression of trans-isoprenyl diphosphate synthase (IDS), the key enzyme involved in the biosynthesis of trans-polyisoprene. trans-Polyisoprene is a particularly valuable compound due to its superior stiffness, excellent insulation, and low thermal expansion coefficient. Currently, trans-polyisoprene is mainly produced through chemical synthesis and no biotechnological processes have been established so far for its large-scale production. In this work, we employed D-optimal design and response surface methodology to optimize the expression of thermophilic enzymes IDS from Thermococcus kodakaraensis. The design of experiment took into account of six factors (preinduction cell density, inducer concentration, postinduction temperature, salt concentration, alternative carbon source, and protein inhibitor) and seven culture media (LB, NZCYM, TB, M9, Ec, Ac, and EDAVIS) at five different pH points. By screening only 109 experimental points, we were able to improve IDS production by 48% in close-batch fermentation. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

δ-Deuterium Isotope Effects as Probes for Transition-State Structures of Isoprenoid Substrates

PubMed Central

2015-01-01

The biosynthetic pathways to isoprenoid compounds involve transfer of the prenyl moiety in allylic diphosphates to electron-rich (nucleophilic) acceptors. The acceptors can be many types of nucleophiles, while the allylic diphosphates only differ in the number of isoprene units and stereochemistry of the double bonds in the hydrocarbon moieties. Because of the wide range of nucleophilicities of naturally occurring acceptors, the mechanism for prenyltransfer reactions may be dissociative or associative with early to late transition states. We have measured δ-secondary kinetic isotope effects operating through four bonds for substitution reactions with dimethylallyl derivatives bearing deuterated methyl groups at the distal (C3) carbon atom in the double bond under dissociative and associative conditions. Computational studies with density functional theory indicate that the magnitudes of the isotope effects correlate with the extent of bond formation between the allylic moiety and the electron-rich acceptor in the transition state for alkylation and provide insights into the structures of the transition states for associative and dissociative alkylation reactions. PMID:24665882

Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions

PubMed Central

Thai, Long; Rush, Jeffrey S.; Maul, Jude E.; Devarenne, Timothy; Rodgers, Dana L.; Chappell, Joseph; Waechter, Charles J.

1999-01-01

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [3H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [3H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [3H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [3H]F-OH or [3H]geranylgeraniol ([3H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [3H]F-P and [3H]F-P-P were synthesized when exogenous [3H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor–product relationship between [3H]F-P and [3H]F-P-P. In agreement with this kinetic pattern of labeling, [32P]F-P and [32P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [γ-32P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [3H]GG-OH to [3H]geranylgeranyl monophosphate and [3H]geranylgeranyl pyrophosphate ([3H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [3H]CTP was formed when microsomes were incubated with [3H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation. PMID:10557276

Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice via suppressing aldo-keto reductase family 1B10 (AKR1B10).

PubMed

Li, Haonan; Yang, Allison L; Chung, Yeon Tae; Zhang, Wanying; Liao, Jie; Yang, Guang-Yu

2013-09-01

Sulindac has been identified as a competitive inhibitor of aldo-keto reductase 1B10 (AKR1B10), an enzyme that plays a key role in carcinogenesis. AKR1B10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl. There have been no studies though showing that the inhibition of PDAC by sulindac is via inhibition of AKR1B10, particularly the metabolism of farnesyl/geranylgeranyl and Kras protein prenylation. To determine the chemopreventive effects of sulindac on pancreatic carcinogenesis, 5-week-old LSL-Kras(G12D)-LSL-Trp53(R172H)-Pdx-1-Cre mice (Pan(kras/p53) mice) were fed an AIN93M diet with or without 200 p.p.m. sulindac (n = 20/group). Kaplan-Meier survival analysis showed that average animal survival in Pan(kras/p53) mice was 143.7 ± 8.8 days, and average survival with sulindac was increased to 168.0 ± 8.8 days (P < 0.005). Histopathological analyses revealed that 90% of mice developed PDAC, 10% with metastasis to the liver and lymph nodes. With sulindac, the incidence of PDAC was reduced to 56% (P < 0.01) and only one mouse had lymph node metastasis. Immunochemical analysis showed that sulindac significantly decreased Ki-67-labeled cell proliferation and markedly reduced the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Raf and mitogen-activated protein kinase kinase 1 and 2. In in vitro experiments with PDAC cells from Pan(kras/p53) mice, sulindac exhibited dose-dependent inhibition of AKR1B10 activity. By silencing AKR1B10 expression through small interfering RNA or by sulindac treatment, these in vitro models showed a reduction in Kras and human DNA-J homolog 2 protein prenylation, and downregulation of phosphorylated C-raf, ERK1/2 and MEK1/2 expression. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis by the inhibition of Kras protein prenylation by targeting AKR1B10.

Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions.

PubMed

Thai, L; Rush, J S; Maul, J E; Devarenne, T; Rodgers, D L; Chappell, J; Waechter, C J

1999-11-09

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [(3)H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [(3)H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [(3)H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [(3)H]F-OH or [(3)H]geranylgeraniol ([(3)H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [(3)H]F-P and [(3)H]F-P-P were synthesized when exogenous [(3)H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor-product relationship between [(3)H]F-P and [(3)H]F-P-P. In agreement with this kinetic pattern of labeling, [(32)P]F-P and [(32)P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [gamma-(32)P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [(3)H]GG-OH to [(3)H]geranylgeranyl monophosphate and [(3)H]geranylgeranyl pyrophosphate ([(3)H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [(3)H]CTP was formed when microsomes were incubated with [(3)H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.

Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide-sensitive K-channel in smooth muscle cells of rat mesenteric artery.

PubMed Central

Zhang, H; Bolton, T B

1995-01-01

1. Single-channel recordings were made from cell-attached and isolated patches, and whole-cell currents were recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery. 2. In single voltage-clamped cells 1 mM uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 microM) a K-channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 microM). In single cells from which recordings were made by the 'perforated patch' (nystatin pipette) technique, metabolic inhibition by 1 mM NaCN and 10 mM 2-deoxy-glucose also evoked a similar glibenclamide-sensitive current. 3. Single K-channel activity was observed in cell-attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide-sensitive channel activity. If the patch was pulled off the cell to form an isolated inside-out patch, similar glibenclamide-sensitive single-channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell-attached mode; channel conductance was 20 pS (60:130 K-gradient) and openings showed no voltage-dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K-channel (KNDP) seen previously in rabbit portal vein. 4. Formation of an isolated inside-out patch into an ATP-free solution did not increase the probability of channel opening which declined with time even when some single-channel activity had occurred in the cell-attached mode before detachment. However, application of 1 mM UDP or GDP, but not ATP, to inside-out patches evoked single-channel activity. Application of ATP-free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke channel activity. 5. It is concluded that small conductance K-channels (KNDP) open in smooth muscle cells from this small artery in response to UDP or GDP acting from the inside, or pinacidil acting from the outside; the same channels open during inhibition of metabolism presumably mainly due to the rise in nucleoside diphosphates, but a fall in the ATP concentration on the inside of the channel did not by itself evoke channel activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7735693

Electron attachment to DNA single strands: gas phase and aqueous solution

PubMed Central

Gu, Jiande; Xie, Yaoming; Schaefer, Henry F.

2007-01-01

The 2′-deoxyguanosine-3′,5′-diphosphate, 2′-deoxyadenosine-3′,5′-diphosphate, 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3′,5′-dTDP (0.17 eV) and 3′,5′-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3′,5′-dTDP > 3′,5′-dCDP > 3′,5′-dGDP > 3′,5′-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3′-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3′-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3′,5′-dADP− (0.26 eV) and 3′,5′-dGDP− (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers such as glycosidic bond breakage. However, the radical anions of the pyrimidine nucleotides with high VDE are expected to be electronically stable. Thus the base-centered radical anions of the pyrimidine nucleotides might be the possible intermediates for DNA single-strand breakage. PMID:17660189

Transcription profile data of phorbol esters biosynthetic genes during developmental stages in Jatropha curcas.

PubMed

Jadid, Nurul; Mardika, Rizal Kharisma; Purwani, Kristanti Indah; Permatasari, Erlyta Vivi; Prasetyowati, Indah; Irawan, Mohammad Isa

2018-06-01

Jatropha curcas is currently known as an alternative source for biodiesel production. Beside its high free fatty acid content, J. curcas also contains typical diterpenoid-toxic compounds of Euphorbiaceae plant namely phorbol esters. This article present the transcription profile data of genes involved in the biosynthesis of phorbol esters at different developmental stages of leaves, fruit, and seed in Jatropha curcas . Transcriptional profiles were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). We used two genes including GGPPS (Geranylgeranyl diphospate synthase), which is responsible for the formation of common diterpenoid precursor (GGPP) and CS (Casbene Synthase), which functions in the synthesis of casbene. Meanwhile, J. curcas Actin ( ACT ) was used as internal standard. We demonstrated dynamic of GGPPS and CS expression among different stage of development of leaves, fruit and seed in Jatropha .

Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction.

PubMed

Cao, Xuan; Lv, Yu-Bei; Chen, Jun; Imanaka, Tadayuki; Wei, Liu-Jing; Hua, Qiang

2016-01-01

Limonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene. To construct the limonene synthetic pathway in Yarrowia lipolytica , two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica . Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW. This is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes.

Gene cloning, expression, and characterization of trehalose-6-phosphate synthase from Pleurotus ostreatus.

PubMed

Lei, Min; Wu, Xiangli; Zhang, Jinxia; Wang, Hexiang; Huang, Chenyang

2017-07-01

Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) catalyzes the first step in trehalose synthesis, which involves transfer of glucose from uridine diphosphate glucose (UDPG) to glucose 6-phosphate (G6P) to form trehalose-6-phosphate. To determine the gene and enzymatic characteristics of TPS in Pleurotus ostreatus, we cloned and sequenced the cDNA of PoTPS1, which contains a 1665 bp open reading frame that encodes a 554-amino acid protein with a predicted molecular weight of 62.01 kDa. This gene was expressed in Escherichia coli BL21 and then the recombinant protein was purified and characterized. Results showed that the optimum pH and temperature for the recombinant PoTPS1 were 7.4 and 30 °C, respectively; the K m value against G6P and UDPG were 0.14 and 0.17 mM, respectively, and the V max and K cat values were 91.86 nkat/g and 5.89 s -1 , respectively. Trehalose content was as high as 158.88 mg g -1 dry weight after heat treatment at 40 °C for 15 h, which was consistent with highest TPS1 activity at that time point. This result indicated that PoTPS1 was responsible for trehalose synthesis in P. ostreatus. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

PubMed Central

Haase, Volker H.; King, Rhonda; Polyak, Erzsebet; Selak, Mary; Yudkoff, Marc; Hancock, Wayne W.; Meade, Ray; Saiki, Ryoichi; Lunceford, Adam L.; Clarke, Catherine F.; Gasser, David L.

2008-01-01

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment. PMID:18437205

Biochemical characterization of an isoprene synthase from Campylopus introflexus (heath star moss).

PubMed

Lantz, Alexandra T; Cardiello, Joseph F; Gee, Taylor A; Richards, Michaelin G; Rosenstiel, Todd N; Fisher, Alison J

2015-09-01

Each year, plants emit terragram quantities of the reactive hydrocarbon isoprene (2-methyl-1,3-butadiene) into the earth's atmosphere. In isoprene-emitting plants, the enzyme isoprene synthase (ISPS) catalyzes the production of isoprene from the isoprenoid intermediate dimethylallyl diphosphate (DMADP). While isoprene is emitted from all major classes of land plants, to date ISPSs from angiosperms only have been characterized. Here, we report the identification and initial biochemical characterization of a DMADP-dependent ISPS from the isoprene-emitting bryophyte Campylopus introflexus (heath star moss). The partially-purified C. introflexus ISPS (CiISPS) exhibited a Km for DMADP of 0.37 ± 0.28 mM, a pH optimum of 8.6 ± 0.5, and a temperature optimum of 40 ± 3 °C in vitro. Like ISPSs from angiosperms, the CiISPS required the presence of a divalent cation. However, unlike angiosperm ISPSs, the CiISPS utilized Mn(2+) preferentially over Mg(2+). Efforts are currently underway in our laboratory to further purify the CiISPS and clone the cDNA sequence encoding this novel enzyme. Our discovery of the first bryophyte ISPS paves the way for future studies concerning the evolutionary origins of isoprene emission in land plants and may help generate new bryophyte model systems for physiological and biochemical research on plant isoprene function. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans

PubMed Central

Masoudi, Neda; Fancsalszky, Luca; Pourkarimi, Ehsan; Vellai, Tibor; Alexa, Anita; Reményi, Attila; Gartner, Anton; Mehta, Anil; Takács-Vellai, Krisztina

2013-01-01

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis. PMID:23900546

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway

PubMed Central

Hsiao, Yu-Yun; Tsai, Wen-Chieh; Kuoh, Chang-Sheng; Huang, Tian-Hsiang; Wang, Hei-Chia; Wu, Tian-Shung; Leu, Yann-Lii; Chen, Wen-Huei; Chen, Hong-Hwa

2006-01-01

Background Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis. Results The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. Conclusion This systems biology program combined chemical analysis, genomics and bioinformatics to elucidate the scent biosynthesis pathway and identify the relevant genes. It integrates the forward and reverse genetic approaches to knowledge discovery by which researchers can study non-model plants. PMID:16836766

Synthetic Analogs of Phospholipid Metabolites as Antimalarials.

DTIC Science & Technology

1979-07-01

phosphatidic acid analogs containing ether and phosphonate groups; completely non-hydrolyzable lecithin analogs containing phosphinate and ether groups...The phosphatidic acid and lecithin target compounds were successfully synthesized and submitted, together with a number of intermediates. A model of a... Phosphatidic acid analogs ......................... 5 Z.Z Lecithin Analogs .................................. 6 2.3 Analogs of Cytidine Diphosphate

The effect of Mg/2+/ and Ca/2+/ on urea-catalyzed phosphorylation reactions

NASA Technical Reports Server (NTRS)

Handschuk, G. J.; Lohrmann, R.; Orgel, L. E.

1973-01-01

The effect of Mg(2+) and Ca(2+) on phosphorylation reactions catalyzed by urea is investigated, showing that Mg(2+) improves markedly the yield of products containing pyrophosphate bonds. Yields of up to 25% of uridine diphosphate can be obtained with struvite at temperatures as low as 65 C.

Production of trichodiene by Trichoderma harzianum alters the perception of this biocontrol strain by plants and antagonized fungi

USDA-ARS?s Scientific Manuscript database

Trichothecenes are phytotoxic sesquiterpenoid compounds of fungal origin which can act as virulence factors in plant diseases. Harzianum A (HA) is a non-phytotoxic trichothecene produced by Trichoderma arundinaceum. The first step in the biosynthesis of HA is the conversion of farnesyl diphosphate t...

Heat Stress Response in Pea Involves Interaction of Mitochondrial Nucleoside Diphosphate Kinase with a Novel 86-Kilodalton Protein1

PubMed Central

Escobar Galvis, Martha L.; Marttila, Salla; Håkansson, Gunilla; Forsberg, Jens; Knorpp, Carina

2001-01-01

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein. PMID:11351071

Identification of a Plastid-Localized Bifunctional Nerolidol/Linalool Synthase in Relation to Linalool Biosynthesis in Young Grape Berries

PubMed Central

Zhu, Bao-Qing; Cai, Jian; Wang, Zhi-Qun; Xu, Xiao-Qing; Duan, Chang-Qing; Pan, Qiu-Hong

2014-01-01

Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS) synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and nerolidol in vitro, respectively, and thus it was named VvRILinNer. However, localization experiment showed that this enzyme was only localized to chloroplasts, which indicates that VvRILinNer functions in the linalool production in vivo. The patterns of gene expression and linalool accumulation were analyzed in the berries of three grape cultivars (“Riesling”, “Cabernet Sauvignon”, “Gewurztraminer”) with significantly different levels of monoterpenoids. The VvRILinNer was considered to be mainly responsible for the synthesis of linalool at the early developmental stage. This finding has provided us with new knowledge to uncover the complex monoterpene biosynthesis in grapes. PMID:25470020

Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity in Vitro and in Vivo against Vancomycin-Resistant Enterococci.

PubMed

Mohammad, Haroon; Younis, Waleed; Chen, Lu; Peters, Christine E; Pogliano, Joe; Pogliano, Kit; Cooper, Bruce; Zhang, Jianan; Mayhoub, Abdelrahman; Oldfield, Eric; Cushman, Mark; Seleem, Mohamed N

2017-03-23

The emergence of antibiotic-resistant bacterial species, such as vancomycin-resistant enterococci (VRE), necessitates the development of new antimicrobials. Here, we investigate the spectrum of antibacterial activity of three phenylthiazole-substituted aminoguanidines. These compounds possess potent activity against VRE, inhibiting growth of clinical isolates at concentrations as low as 0.5 μg/mL. The compounds exerted a rapid bactericidal effect, targeting cell wall synthesis. Transposon mutagenesis suggested three possible targets: YubA, YubB (undecaprenyl diphosphate phosphatase (UPPP)), and YubD. Both UPPP as well as undecaprenyl diphosphate synthase were inhibited by compound 1. YubA and YubD are annotated as transporters and may also be targets because 1 collapsed the proton motive force in membrane vesicles. Using Caenorhabditis elegans, we demonstrate that two compounds (1, 3, at 20 μg/mL) retain potent activity in vivo, significantly reducing the burden of VRE in infected worms. Taken altogether, the results indicate that compounds 1 and 3 warrant further investigation as novel antibacterial agents against drug-resistant enterococci.

Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein.

PubMed

Escobar Galvis, M L; Marttila, S; Håkansson, G; Forsberg, J; Knorpp, C

2001-05-01

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein.

Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography.

PubMed

Lima, Juliana Maria; Salmazo Vieira, Plínio; Cavalcante de Oliveira, Arthur Henrique; Cardoso, Carmen Lúcia

2016-08-07

Nucleoside diphosphate kinase from Leishmania spp. (LmNDKb) has recently been described as a potential drug target to treat leishmaniasis disease. Therefore, screening of LmNDKb ligands requires methodologies that mimic the conditions under which LmNDKb acts in biological systems. Here, we compare two label-free methodologies that could help screen LmNDKb ligands and measure NDKb activity: an offline LC-UV assay for soluble LmNDKb and an online two-dimensional LC-UV system based on LmNDKb immobilised on a silica capillary. The target enzyme was immobilised on the silica capillary via Schiff base formation (to give LmNDKb-ICER-Schiff) or affinity attachment (to give LmNDKb-ICER-His). Several aspects of the ICERs resulting from these procedures were compared, namely kinetic parameters, stability, and procedure steps. Both the LmNDKb immobilisation routes minimised the conformational changes and preserved the substrate binding sites. However, considering the number of steps involved in the immobilisation procedure, the cost of reagents, and the stability of the immobilised enzyme, immobilisation via Schiff base formation proved to be the optimal procedure.

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli.

PubMed

Mendez-Perez, Daniel; Alonso-Gutierrez, Jorge; Hu, Qijun; Molinas, Margaux; Baidoo, Edward E K; Wang, George; Chan, Leanne J G; Adams, Paul D; Petzold, Christopher J; Keasling, Jay D; Lee, Taek S

2017-08-01

Monoterpenes (C 10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C 15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of Escherichia coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP production but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes. Biotechnol. Bioeng. 2017;114: 1703-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Utilization of biodiesel by-product as substrate for high-production of β-farnesene via relatively balanced mevalonate pathway in Escherichia coli.

PubMed

You, Shengping; Yin, Qingdian; Zhang, Jianye; Zhang, Chengyu; Qi, Wei; Gao, Lan; Tao, Zhiping; Su, Rongxin; He, Zhimin

2017-11-01

Farnesene has been identified as suitable jet fuel substitutes and metabolic engineering for microbial production of farnesene is an alternative and attractive route. In this study, due to accumulation of toxic intermediate isopentenyl pyrophosphate (IPP), an engineered Escherichia coli strain harboring heterologous mevalonate pathway produced only 4.11mg/L β-farnesene. Through higher-level expression of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase to minimize the accumulated IPP, another engineered strain with relatively balanced mevalonate pathway was constructed and had the highest production of β-farnesene to date (8.74g/L) by Escherichia coli in a lab bioreactor. Furthermore, this is the first report on utilization of biodiesel by-product (simple purification) as substrate for high-production of β-farnesene by the engineered strain optimized and β-farnesene concentration reached 2.83g/L in a lab bioreactor. Therefore, the engineered strain optimized could be used as a platform host for high-production of other terpenoids using biodiesel by-product as substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

PubMed

Wen, Zewen; Liu, Zhiyong; Hou, Yuyong; Liu, Chenfeng; Gao, Feng; Zheng, Yubin; Chen, Fangjian

2015-10-01

Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis. Copyright © 2015 Elsevier Inc. All rights reserved.

Biosynthesis of yeast glycoproteins. Processing of the oligosaccharides transferred from dolichol derivatives.

PubMed

Parodi, A J

1979-10-25

The oligosaccharides previously bound to dolichol diphosphate were isolated from Saccharomyces cerevisiae cells incubated with [U-14C]glucose. Five compounds were obtained that migrated with RGlucose of 0.100, 0.120, 0.145, 0.180, and 0.215 on paper chromatography. All of them contained mannose and 2 N-acetylhexosamine residues. The substances that migrated with the three lower RGlucose values had, in addition, glucose units. The structure of the oligosacchardies was very similar if not identical with that of the oligosaccharides isolated from the dolichol diphosphate derivatives synthesized "in vitro" by yeast or rat liver particulate preparations or "in vivo" by dog thyroid or rat liver slices as judged by their migration on paper chromatography, monosaccharide composition, and degradation compounds produced by alpha-mannosidase treatment or acetolysis. The oligosaccharides previously bound to asparagine residues in proteins were isolated from yeast cells which had been pulsed with [U-14C]glucose and chased with medium containing the unlabeled monosaccharide. The samples taken after very short pulses contained four oligosaccharides that migrated with RGlucose of 0.100, 0.120, 0.145, and 0.180 on paper chromatography. The first three compounds contained glucose, mannose, and 2 N-acetylhexosamine residues whereas the one that migrated with a RGlucose of 0.180 was devoid of the former monosaccharide. Samples taken after short chase periods revealed that the compounds that migrated with the lower RGlucose values gradually disappeared and were converted to the oligosaccharide with the higher RGlucose value was they lost their glucose residues. Similar analysis as those mentioned above showed that the structures of these compounds were similar to those of the dolichol diphosphate-bound oligosaccharides. Samples taken after longer chase periods revealed that the oligosaccharide that migrated with a RGlucose of 0.180 was subsequently either enlarged by the addition of more mannose residues or trimmed to smaller sizes.

Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

PubMed Central

Peng, Qian-Yi; Zou, Yu; Zhang, Li-Na; Ai, Mei-Lin; Liu, Wei; Ai, Yu-Hang

2016-01-01

Background: Acute lung injury (ALI) is a common complication of sepsis that is associated with high mortality. Intracellular Ca2+ overload plays an important role in the pathophysiology of sepsis-induced ALI, and cyclic adenosine diphosphate ribose (cADPR) is an important regulator of intracellular Ca2+ mobilization. The cluster of differentiation 38 (CD38)/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALI is still unknown. This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALI and whether blocking cADPR-mediated calcium overload attenuates ALI. Methods: Septic rat models were established by cecal ligation and puncture (CLP). Rats were divided into the sham group, the CLP group, and the CLP+ 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR) group. Nicotinamide adenine dinucleotide (NAD+), cADPR, CD38, and intracellular Ca2+ levels in the lung tissues were measured at 6, 12, 24, and 48 h after CLP surgery. Lung histologic injury, tumor necrosis factor (TNF)-α, malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activities were measured. Results: NAD+, cADPR, CD38, and intracellular Ca2+ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery. Treatment with 8-Br-cADPR, a specific inhibitor of cADPR, significantly reduced intracellular Ca2+ levels (P = 0.007), attenuated lung histological injury (P = 0.023), reduced TNF-α and MDA levels (P < 0.001 and P = 0.002, respectively) and recovered SOD activity (P = 0.031) in the lungs of septic rats. Conclusions: The CD38/cADPR pathway is activated in the lungs of septic rats, and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALI. PMID:27411462

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

PubMed

Ge, Deyong; Xue, Yanfen; Ma, Yanhe

2016-05-11

Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene. Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain. IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

PubMed

Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

2006-06-02

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Comparison of increased aspirin dose versus combined aspirin plus clopidogrel therapy in patients with diabetes mellitus and coronary heart disease and impaired antiplatelet response to low-dose aspirin.

PubMed

Duzenli, Mehmet Akif; Ozdemir, Kurtulus; Aygul, Nazif; Soylu, Ahmet; Tokac, Mehmet

2008-08-15

The effects of therapy with aspirin 300 mg/day and with combined aspirin 100 mg/day plus clopidogrel 75 mg/day on platelet function were compared in patients with diabetes mellitus and coronary artery disease and impaired antiplatelet responses to aspirin 100 mg/day. The study population consisted of 151 outpatients with type II diabetes mellitus and coronary artery disease who were taking aspirin 100 mg/day. Of the 151 patients, a subgroup of subjects with impaired aspirin response were selected on the basis of the results of platelet aggregometry. Nonresponsiveness to aspirin was defined as mean aggregation > or =69% with 3 micromol/L adenosine diphosphate and mean aggregation > or =70% with 2 micromol/L collagen. Aspirin semiresponders were defined as meeting 1 but not both of these criteria. Nonresponders and semiresponders were randomized equally to aspirin 300 mg/day and aspirin 100 mg/day plus clopidogrel 75 mg/day, and aggregation tests were repeated after 2 weeks. Sixty of the 151 patients with diabetes (40%) were found to respond to aspirin inadequately. Platelet aggregation induced by adenosine diphosphate and collagen decreased significantly after aspirin 300 mg/day or combined therapy. Combined treatment was found to have a stronger inhibitory effect on platelet aggregation induced by adenosine diphosphate than aspirin 300 mg/day (p = 0.002). Impaired aspirin response was resolved by increasing the aspirin dose or adding clopidogrel to aspirin (p <0.0001 for each). However, desired platelet inhibition was achieved in significantly more patients by combined treatment than by aspirin 300 mg/day (p <0.05). In conclusion, aspirin 100 mg/day does not inhibit platelet function adequately in a significant number of patients with diabetes mellitus and coronary artery disease. Increasing the aspirin dose to 300 mg/day or adding clopidogrel to aspirin can provide adequate platelet inhibition in a significant number of those patients with impaired responses to low-dose aspirin.

Assessment of platelet function in healthy cats in response to commonly prescribed antiplatelet drugs using three point-of-care platelet function tests.

PubMed

Ho, Kimberly K; Abrams-Ogg, Anthony Cg; Wood, R Darren; O'Sullivan, M Lynne; Kirby, Gordon M; Blois, Shauna L

2017-06-01

Objectives The objective was to determine if decreased platelet function could be detected after treatment with aspirin and/or clopidogrel in healthy cats using three point-of-care platelet function tests that evaluate platelet function by different methods: Multiplate (by impedance), Platelet Function Analyzer 100 (by mechanical aperture closure) and Plateletworks (by platelet counting). Methods Thirty-six healthy cats were randomly assigned to receive one of three oral treatments over an 8 day period: (1) aspirin 5 mg q72h; (2) aspirin 20.25 mg q72h; or (3) clopidogrel 18.75 mg q24h. Cats treated with 5 and 20.25 mg aspirin also received clopidogrel on days 4-8. Platelet aggregation in response to adenosine diphosphate and collagen ± arachidonic acid was assessed on days 1 (baseline), 4 and 8. Aspirin and clopidogrel metabolites were measured by high-performance liquid chromatography. Platelet function in response to treatment was analyzed by ANCOVA, linear regression and Spearman correlation. Results The only solitary aspirin effect was detected using Plateletworks with collagen in cats treated with 20.25 mg. The only effect detected by Multiplate was using arachidonic acid in cats treated with both aspirin 20.25 mg and clopidogrel. All clopidogrel treatment effects were detected by Platelet Function Analyzer 100, Plateletworks (adenosine diphosphate) and Plateletworks (collagen). Drug metabolites were present in all cats, but concentrations were minimally correlated to platelet function test results. Conclusions and relevance Platelet Function Analyzer 100 and Plateletworks using adenosine diphosphate ± collagen agonists may be used to detect decreased platelet function in response to clopidogrel treatment. Either aspirin is not as effective an antiplatelet drug as clopidogrel, or the tests used were not optimal to measure aspirin effect. Cats with heart disease are commonly prescribed antiplatelet drugs to decrease the risk of aortic thromboembolism. Platelet Function Analyzer 100 and Plateletworks may be useful for confirming clopidogrel treatment in these cats.

Tetraether bolaform amphiphiles as models of archaebacterial membrane lipids: Synthesis, differential scanning calorimetry, and monolayer studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, J.M.; Thompson, D.H.

Four racemic tetraether lipids containing a single 1,[omega]-polymethylene chain ([omega] = 16, 20) bridging two glycerophosphate headgroups (bolaform amphiphiles) have been synthesized. These materials have been characterized at the air-water interface by monolayer balance methods and in buffered solution by differential scanning calorimetry (DSC) and negative stain transmission electron microscopy (TEM). Molecular areas in excess of 100 [angstrom][sup 2]/molecule at 40 mN/m[sup 2] were observed for all bolaamphiphiles studied, suggesting a U-shaped molecular conformation that places both phosphate headgroups in the water subphase. Aqueous dispersions of these lipids have thermal and morphological properties that depend on molecular structure and solutionmore » pH. Phase transition temperatures (T[sub c]) of the structural isomers, 2,2[prime]-di-O-decyl-1, 1[prime]-O-eicosamethylene-rac-diglycero-3,3[prime]-diphosphate (PS20) and 1,1[prime]-di-O-decyl-2,2[prime]-O-eicosamethylene-3,3[prime]-diphosphate (SS20), were 49 and 38 [degrees]C, respectively, at pH 2.5. A reduction in the observed T[sub c] of [approximately] 14 [degrees]C occurred when the pH was raised to 8.1. The closely related structural analogue, 1,1[prime]-O-eicosamethylene-2-O-eicosyl-rac-diglycero-3,2[prime], 3[prime]-diphosphate (PA20), has a T[sub c] 85 [degrees]C. No phase transition was observed above 5 [degrees]C for 2,2[prime]-O-dioctyl-1,1 [prime]-O-hexadecylmethylene-rac-diglycero-3, 3[prime]-disphosphoric acid (PS16). Multilamellar structures with hydrocarbon-region spacings of 24-30 [angstrom] and overall lengths approaching 0.3 [mu]m were observed by negative stain electron microscopy. The observed lamellae distance is in good agreement with the membrane thickness expected for a bolaamphiphile in its all-anti conformation. 56 refs., 8 figs., 1 tab.« less

Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis

PubMed Central

Jiang, Xiao-Sheng; Backlund, Peter S.; Wassif, Christopher A.; Yergey, Alfred L.; Porter, Forbes D.

2010-01-01

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7Δ3–5/Δ3–5 and Sc5d−/− E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7Δ3–5/Δ3–5 and Sc5d−/− brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7Δ3–5/Δ3–5 and Sc5d−/− embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis. PMID:20305089

Protein prenylation: a new mode of host-pathogen interaction.

PubMed

Amaya, Moushimi; Baranova, Ancha; van Hoek, Monique L

2011-12-09

Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction. Copyright © 2011 Elsevier Inc. All rights reserved.

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

PubMed

Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

2000-02-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

Limonene Arrests Parasite Development and Inhibits Isoprenylation of Proteins in Plasmodium falciparum

PubMed Central

Moura, Ivan Cruz; Wunderlich, Gerhard; Uhrig, Maria L.; Couto, Alicia S.; Peres, Valnice J.; Katzin, Alejandro M.; Kimura, Emília A.

2001-01-01

Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development. PMID:11502528

Structure of the Integral Membrane Protein CAAX Protease Ste24p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pryor Jr., Edward E.; Horanyi, Peter S.; Clark, Kathleen M.

2012-10-26

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavitymore » containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.« less

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

PubMed Central

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

In silico identification and analysis of phytoene synthase genes in plants.

PubMed

Han, Y; Zheng, Q S; Wei, Y P; Chen, J; Liu, R; Wan, H J

2015-08-14

In this study, we examined phytoene synthetase (PSY), the first key limiting enzyme in the synthesis of carotenoids and catalyzing the formation of geranylgeranyl pyrophosphate in terpenoid biosynthesis. We used known amino acid sequences of the PSY gene in tomato plants to conduct a genome-wide search and identify putative candidates in 34 sequenced plants. A total of 101 homologous genes were identified. Phylogenetic analysis revealed that PSY evolved independently in algae as well as monocotyledonous and dicotyledonous plants. Our results showed that the amino acid structures exhibited 5 motifs (motifs 1 to 5) in algae and those in higher plants were highly conserved. The PSY gene structures showed that the number of intron in algae varied widely, while the number of introns in higher plants was 4 to 5. Identification of PSY genes in plants and the analysis of the gene structure may provide a theoretical basis for studying evolutionary relationships in future analyses.

Ophirapstanol trisulfate, a new biologically active steroid sulfate from the deep water marine sponge Topsentia ophiraphidites.

PubMed

Gunasekera, S P; Sennett, S H; Kelly-Borges, M; Bryant, R W

1994-12-01

Ophirapstanol trisulfate [1], a new steroid trisulfate related to sokotrasterol trisulfate was isolated from a deep water marine sponge Topsentia ophiraphidites. Compound 1 exhibited significant inhibition in the guanosine diphosphate/G-protein RAS exchange assay. The structure elucidation of 1 and ophirapstanol [2] by nmr spectroscopy is described.

Problem-solving test: catalytic activities of a human nuclear enzyme.

PubMed

Szeberényi, József

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5′-end and 3′-end, bacteriophage, polyacrylamide gel electrophoresis, urea, autoradiography, proofreading, telomerase, endonucleases, exonucleases, primase, topoisomerases, and excinuclease.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide

EPA Science Inventory

An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosph...

Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation 1

PubMed Central

Vasconcelos, Aurea; Pollack, Marilyn; Mendiola, Leticia R.; Hoffmann, H.-P.; Brown, D. H.; Price, C. A.

1971-01-01

Chloroplasts were separated from Euglena gracilis by zonal centrifugation at low speed in density gradients of Ficoll or dextran. The chloroplasts were intact by the criteria of ultrastructure and their content of ribulose diphosphate carboxylase and soluble protein. The chloroplasts also contained ribosomes and ribosomal RNA uncontaminated by the corresponding cytoplasmic particles. Images PMID:16657599

Stepwise synthesis of oligonucleotides. XXII. The synthesis of Tpsi-loop fragments of yeast tRNAIVal and their analogs.

PubMed Central

Zhenodarova, S M; Klyagina, V P; Smolyaninova, O A; Khabarova, M I; Antonovich, E G; Prokof'yev, M A

1977-01-01

The method of the combined use of nucleolytic enzymes was used for the synthesis of Tpsi-loop fragments of yeast valine tRNA and their analogs. Dinucleoside monophosphates, trinucleoside diphosphates and tetranucleoside triphosphates having the sequences of fragments 54-57 and 59-62 or their analogs were synthesized. PMID:896487

Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance | Poster

Cancer.gov

Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

Involvement of nucleotide diphosphate kinase 2 in the reopening of the sensitive period of filial imprinting of domestic chicks (Gallus gallus domesticus).

PubMed

Yamaguchi, Shinji; Aoki, Naoya; Takehara, Akihiko; Mori, Masaru; Kanai, Akio; Matsushima, Toshiya; Homma, Koichi J

2016-01-26

Filial imprinting is a behavior characterized by the sensitive or critical period restricted to the first few days after hatching. Once the sensitive period is closed, it is widely believed that chicks can never be imprinted under natural conditions. Previously, we showed that the exogenous injection of T3 reopened the sensitive period which was already closed. That study suggested that T3 functioned by way of a rapid non-genomic action; however, the molecular mechanism of how T3 reopens the sensitive period remains unknown. Here, we show that the phosphorylation level of nucleotide diphosphate kinase 2 (NDPK2) was upregulated following T3 injection. Pharmacological deprivation of the kinase activity of NDPK hampered the molecular process prerequisite for the reopening of the sensitive period of filial imprinting. Moreover, it is shown that the kinase activity of NDPK2 participates in the priming process by T3 signaling which endows the potential for learning. Our data indicate that NDPK2 plays a crucial role downstream of T3 action and that its phosphorylation is involved in the non-genomic signaling during imprinting. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis.

PubMed

Yamamura, Yoshimi; Kurosaki, Fumiya; Lee, Jung-Bum

2017-03-07

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis

PubMed Central

Yamamura, Yoshimi; Kurosaki, Fumiya; Lee, Jung-Bum

2017-01-01

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis. PMID:28266568

Carbon Dioxide Metabolism in Leaf Epidermal Tissue 1

PubMed Central

Willmer, C. M.; Pallas, J. E.; Black, C. C.

1973-01-01

A number of plant species were surveyed to obtain pure leaf epidermal tissue in quantity. Commelina communis L. and Tulipa gesnariana L. (tulip) were chosen for further work. Chlorophyll a/b ratios of epidermal tissues were 2.41 and 2.45 for C. communis and tulip, respectively. Phosphoenolpyruvate carboxylase, ribulose-1,5-diphosphate carboxylase, malic enzyme, and NAD+ and NADP+ malate dehydrogenases were assayed with epidermal tissue and leaf tissue minus epidermal tissue. In both species, there was less ribulose 1,5-diphosphate than phosphoenolpyruvate carboxylase activity in epidermal tissue whether expressed on a protein or chlorophyll basis whereas the reverse was true for leaf tissue minus epidermal tissue. In both species, malic enzyme activities were higher in epidermal tissue than in the remaining leaf tissue when expressed on a protein or chlorophyll basis. In both species, NAD+ and NADP+ malate dehydrogenase activities were higher in the epidermal tissue when expressed on a chlorophyll basis; however, on a protein basis, the converse was true. Microautoradiography of C. communis epidermis and histochemical tests for keto acids suggested that CO2 fixation occurred predominantly in the guard cells. The significance and possible location of the enzymes are discussed in relation to guard cell metabolism. Images PMID:16658581

Stage-specific regulation of juvenile hormone biosynthesis by ecdysteroid in Bombyx mori.

PubMed

Kaneko, Yu; Kinjoh, Terunori; Kiuchi, Makoto; Hiruma, Kiyoshi

2011-03-30

In the penultimate (4th) instar larvae of Bombyx mori, juvenile hormone (JH) synthesis by corpora allata (CA) fluctuates. When diet containing 20-hydroxyecdysone (20E) was fed, JH synthetic activity of the CA was first stimulated as the ecdysteroid titer increased, then suppressed slightly by the higher molting concentration of ecdysteroids (>250 ng/ml). The overall JH biosynthetic activity was modulated by the expression of JH biosynthetic enzymes in the CA: primarily JH acid O-methyltransferase (JHAMT), isopentenyl diphosphate isomerase, and farnesyl diphosphate synthase 1. After the last (5th) larval ecdysis, the artificially increased high ecdysteroid level due to the 20E diet activated JH synthesis by the CA, which required intact nervous connections with the brain. A factor(s) from the 20E-activated brain controls mainly JHAMT and HMG Co-A reductase expression to stimulate the JH synthesis. In the normal last instar larvae, the ecdysteroid titer declines so that these activation mechanisms are absent; therefore the decline of the ecdysteroid titer after the final larval ecdysis is one of the factors which induces the cessation of the JH synthesis by CA. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Carbohydrate metabolism changes in Prunus persica gummosis infected with Lasiodiplodia theobromae.

PubMed

Li, Z; Gao, L; Wang, Y T; Zhu, W; Ye, J L; Li, G H

2014-05-01

Peach gummosis represents a significant global disease of stone fruit trees and a major disease in the south peach production area of the Yangtze River of China. In this study, the carbohydrate composition of peach shoots during infection by Lasiodiplodia theobromae was examined. The expression of genes related to metabolic enzymes was also investigated. Control wounded and noninoculated tissue, lesion tissue, and wounded and inoculated surrounding lesion tissue of peach shoots were analyzed. Soluble sugars, glucose, mannose, arabinose, and xylose significantly increased in inoculated tissues of peach shoots compared with control tissues at different times after inoculation. Accumulation of polysaccharides was also observed by section observation and periodic acid Schiff's reagent staining during infection. Analysis using quantitative reverse-transcription polymerase chain reaction revealed that the abundance of key transcripts on the synthesis pathway of uridine diphosphate (UDP)-D-glucuronate, UDP-D-galactose, and UDP-D-arabinose increased but the synthesis of L-galactose and guanosine diphosphate-L-galactose were inhibited. After inoculation, the transcript levels of sugar transport-related genes (namely, SUT, SOT, GMT, and UGT) was induced. These changes in sugar content and gene expression were directly associated with peach gum polysaccharide formation and may be responsible for the symptoms of peach gummosis.

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems.

PubMed

Kemper, Katarina; Hirte, Max; Reinbold, Markus; Fuchs, Monika; Brück, Thomas

2017-01-01

With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli , this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Platelet mapping as part of modified thromboelastography (TEG®) in patients undergoing cardiac surgery and cardiopulmonary bypass.

PubMed

Weitzel, N S; Weitzel, L B; Epperson, L E; Karimpour-Ford, A; Tran, Z V; Seres, T

2012-10-01

The platelet-mapping assay of the thromboelastograph was used to measure platelet aggregation and to examine the effect of cardiopulmonary bypass on multiple platelet receptors and the role of altered receptor activity in postoperative bleeding. The percentage platelet aggregation for collagen, adenosine diphosphate and arachidonic acid was measured in 40 patients divided post-hoc into high- or low-bleeding groups depending on postoperative 24-h chest tube output. Platelet aggregation was lower after cardiopulmonary bypass compared to before it using collagen (mean (SD) 45 (25) vs 19 (12)%, p<0.001), adenosine diphosphate (76 (23) vs 35 (24)%, p<0.001), and arachidonic acid (61 (33) vs 31 (35)%, p<0.001). Only platelet aggregation as measured using collagen pre- and post-cardiopulmonary bypass was significantly less in the high- compared to the low-bleeding group. This finding was significantly correlated with the 24-h chest tube drainage, and it predicted postoperative bleeding with a sensitivity of 83% and a specificity of 68%. Therefore, platelet aggregation is reduced following cardiopulmonary bypass, and this may play a role in predicting postoperative blood loss. Anaesthesia © 2012 The Association of Anaesthetists of Great Britain and Ireland.

Progranulin inhibits platelet aggregation and prolongs bleeding time in rats.

PubMed

Al-Yahya, A M; Al-Masri, A A; El Eter, E A; Hersi, A; Lateef, R; Mawlana, O

2018-05-01

Several adipokines secreted by adipose tissue have an anti-thrombotic and anti-atherosclerotic function. Recently identified adipokine progranulin was found to play a protective role in atherosclerosis. Bearing in mind the central role of platelets in inflammation and atherosclerosis, we aimed, in this study, to examine the effect of progranulin on platelet function and coagulation profile in rats. Healthy male albino Wistar rats weighing (250-300 g) were divided into 4 groups. Three groups were given increasing doses of progranulin (0.001 µg, 0.01 µg, and 0.1 µg) intraperitoneally, while the control group received phosphate-buffered saline (PBS). Bleeding time, prothrombin time, activated partial thromboplastin time and platelet aggregation responses to adenosine diphosphate and arachidonic acid were assessed. Administration of progranulin resulted in a significant inhibition of platelet aggregation in response to both adenosine diphosphate, and arachidonic acid. Bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in all groups that received progranulin, in particular, the 0.1 µg dose, in comparison to the control group. This preliminary data is first suggesting that the antiplatelet and anticoagulant action of progranulin could have a physiological protective function against thrombotic disorders associated with obesity and atherosclerosis. However, these results merit further exploration.

Chemical synthesis of guanosine diphosphate mannuronic acid (GDP-ManA) and its C-4-O-methyl and C-4-deoxy congeners.

PubMed

Zhang, Qingju; Howell, P Lynne; Overkleeft, Herman S; Filippov, Dmitri V; van der Marel, Gijsbert A; Codée, Jeroen D C

2017-10-10

Described is the first synthesis of guanosine diphosphate mannuronic acid (GDP-ManA), the sugar donor used by algae and bacteria for the production of alginate, an anionic polysaccharide composed of β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). Understanding the biosynthesis of these polyanionic polysaccharides on the molecular level, opens up avenues to use and modulate the biosynthesis machinery for biotechnological and therapeutic applications. The synthesis reported here delivers multi-milligram amounts of the GDP-ManA donor that can be used to study the polymerase (Alg8 in Pseudomonas aeruginosa) that generates the poly-ManA chain. Also reported is the assembly of two close analogues of GDP-ManA: the first bears a C-4-O-methyl group, while the second has been deoxygenated at this position. Both molecules may be used as "chain stoppers" in future enzymatic ManA polymerisation reactions. The crucial pyrophosphate linkage of the GDP-mannuronic acids has been constructed by the phosphorylation of the appropriate ManA-1-phosphates with a guanosine phosphoramidite. Copyright © 2017 Elsevier Ltd. All rights reserved.

In-silico Leishmania target selectivity of antiparasitic terpenoids.

PubMed

Ogungbe, Ifedayo Victor; Setzer, William N

2013-07-03

Neglected Tropical Diseases (NTDs), like leishmaniasis, are major causes of mortality in resource-limited countries. The mortality associated with these diseases is largely due to fragile healthcare systems, lack of access to medicines, and resistance by the parasites to the few available drugs. Many antiparasitic plant-derived isoprenoids have been reported, and many of them have good in vitro activity against various forms of Leishmania spp. In this work, potential Leishmania biochemical targets of antiparasitic isoprenoids were studied in silico. Antiparasitic monoterpenoids selectively docked to L. infantum nicotinamidase, L. major uridine diphosphate-glucose pyrophosphorylase and methionyl t-RNA synthetase. The two protein targets selectively targeted by germacranolide sesquiterpenoids were L. major methionyl t-RNA synthetase and dihydroorotate dehydrogenase. Diterpenoids generally favored docking to L. mexicana glycerol-3-phosphate dehydrogenase. Limonoids also showed some selectivity for L. mexicana glycerol-3-phosphate dehydrogenase and L. major dihydroorotate dehydrogenase while withanolides docked more selectively with L. major uridine diphosphate-glucose pyrophosphorylase. The selectivity of the different classes of antiparasitic compounds for the protein targets considered in this work can be explored in fragment- and/or structure-based drug design towards the development of leads for new antileishmanial drugs.

Studies of levels of biogenic amines in meat samples in relation to the content of additives.

PubMed

Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

2016-01-01

The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality.

Exploiting the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis and its application in synthesis of glycosides.

PubMed

Dai, Longhai; Li, Jiao; Yao, Peiyuan; Zhu, Yueming; Men, Yan; Zeng, Yan; Yang, Jiangang; Sun, Yuanxia

2017-04-20

Glycosylation is a prominent biological mechanism for structural and functional diversity of natural products. Uridine diphosphate-dependent glycosyltransferases with aglycon promiscuity are generally recognised as effective biocatalysts for glycodiversification of natural products for practical applications. In this study, the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis 168 was explored. Bs-YjiC, with uridine diphosphate glucose (UDPG) as sugar donor, exhibited robust capabilities to glycosylate 19 structurally diverse types of drug-like scaffolds with regio- and stereospecificities and form O-, N- and S-linkage glycosides. Twenty-four glycosides of 17 aglycons were purified from scale-up reactions using Bs-YjiC as a biocatalyst, and their structures were confirmed by nuclear magnetic resonance spectra. Furthermore, a one-pot reaction by coupling Bs-YjiC to sucrose synthase from Arabidopsis thaliana was applied to glycosylate pterostilbene. Without adding the costly UDPG as sugar donor, 9mM (3.8g/L) pterostilbene 4'-O-β-glucoside was obtained by periodic feeding of pterostilbene. These results suggest the aglycon promiscuity of Bs-YjiC and demonstrate its significant application prospect in biosynthesis of valuable natural products. Copyright © 2017 Elsevier B.V. All rights reserved.

GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

NASA Astrophysics Data System (ADS)

Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

1992-11-01

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

PubMed

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

2015-04-14

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation.

PubMed

Kadry, Ashraf A; El-Ganiny, Amira M; Mosbah, Rasha A; Kaminskyj, Susan G W

2018-07-01

Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.

Use of an Escherichia coli Recombinant Producing Thermostable Polyphosphate Kinase as an ATP Regenerator To Produce Fructose 1,6-Diphosphate▿ †

PubMed Central

Iwamoto, Seishi; Motomura, Kei; Shinoda, Yasuharu; Urata, Masaaki; Kato, Junichi; Takiguchi, Noboru; Ohtake, Hisao; Hirota, Ryuichi; Kuroda, Akio

2007-01-01

Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate. PMID:17616610

Investigation of the system ThO 2-NpO 2-P 2O 5. Solid solutions of thorium-neptunium (IV) phosphate-diphosphate

NASA Astrophysics Data System (ADS)

Dacheux, N.; Thomas, A. C.; Brandel, V.; Genet, M.

1998-11-01

Considering that phosphate matrices could be potential candidates for the immobilization of actinides or for the final disposal of the excess plutonium from dismantled nuclear weapons, the chemistry of thorium phosphates has been re-examined. In the ThO 2-P 2O 5 system, the thorium phosphate-diphosphate Th 4(PO 4) 4P 2O 7 (TPD) can be synthesized by wet and dry chemical processes. The substitution of thorium by other tetravalent actinides like uranium or plutonium can be obtained for 0 < x < 3.0 and 0 < x < 1.63, respectively. In this work, we report the chemical conditions of synthesis of thorium-neptunium (IV) phosphate-diphosphate solid solutions Th 4- xNp x(PO 4) 4P 2O 7 (TNPD) with 0 < x < 1.6 from a mixture of thorium and neptunium (IV) nitrates and concentrated phosphoric acid. From the variation of the cell parameters and volume, the maximum substitution of Th 4+ by Np 4+ in the TPD structure is evaluated to 2.08 (which corresponds to about 52 mol% of thorium replaced by neptunium (IV)). The field of existence of solid solutions Th 4- xU- xNp- xPuU xUNp xNpPu xPu(PO 4)4P 2O 7 has been calculated. These solid solutions should be synthesized for 5 xU+7 xNp+9 xPu⩽15. In the NpO 2-P 2O 5 system, the unit cell parameters of Np 2O(PO 4) 2 were refined by analogy with U 2O(PO 4) 2 which crystallographic data have been published recently. For Np 2O(PO 4) 2 the unit cell is orthorhombic with the following cell parameters: a=7.033(2) Å, b=9.024(3) Å, c=12.587(6) Å and V=799(1) Å 3. The unit cell parameter obtained for α-NpP 2O 7 ( a=8.586(1) Å) is in good agreement with those already reported in literature.

Ubiquinone and carotene production in the Mucorales Blakeslea and Phycomyces.

PubMed

Kuzina, Vera; Cerdá-Olmedo, Enrique

2007-10-01

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of beta-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.

Geraniol rescues inflammation in cellular and animal models of mevalonate kinase deficiency.

PubMed

Marcuzzi, Annalisa; Crovella, Sergio; Pontillo, Alessandra

2011-01-01

The inhibition of the mevalonate pathway through genetic defects such as mevalonate kinase deficiency (MKD) or pharmacological drugs such as aminobisphosphonates causes a shortage of intermediate compounds, in particular geranylgeranyl-pyrophosphate (GGPP), which is associated with the consequent augmented IL-1β release in monocytes. Considering that, due to its biochemical structure, isoprenoid geraniol enters the mevalonate pathway and may revert the genetic or pharmacological inhibition, the present study tested isoprenoid geraniol in cellular and animal MKD models obtained through the use of aminobisphosphonate pamidronate. The effect of natural isoprenoid geraniol on bacterial induced-inflammation was evaluated in a monocytic cell line (Raw 264.7) and in Balb/c mice treated with pamidronate. Geraniol diminished the levels of inflammatory markers induced by pamidronate stimuli in vitro and in vivo. Geraniol may be proposed as a novel therapeutic approach for the orphan disease MKD, and may also be considered for the evaluation of possible inflammatory side-effects of aminobisphosphonates.

Farnesyltransferase haplodeficiency reduces neuropathology and rescues cognitive function in a mouse model of Alzheimer disease.

PubMed

Cheng, Shaowu; Cao, Dongfeng; Hottman, David A; Yuan, LiLian; Bergo, Martin O; Li, Ling

2013-12-13

Isoprenoids and prenylated proteins have been implicated in the pathophysiology of Alzheimer disease (AD), including amyloid-β precursor protein metabolism, Tau phosphorylation, synaptic plasticity, and neuroinflammation. However, little is known about the relative importance of the two protein prenyltransferases, farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT), in the pathogenesis of AD. In this study, we defined the impact of deleting one copy of FT or GGT on the development of amyloid-β (Aβ)-associated neuropathology and learning/memory impairments in APPPS1 double transgenic mice, a well established model of AD. Heterozygous deletion of FT reduced Aβ deposition and neuroinflammation and rescued spatial learning and memory function in APPPS1 mice. Heterozygous deletion of GGT reduced the levels of Aβ and neuroinflammation but had no impact on learning and memory. These results document that farnesylation and geranylgeranylation play differential roles in AD pathogenesis and suggest that specific inhibition of protein farnesylation could be a potential strategy for effectively treating AD.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

PubMed Central

2011-01-01

Abstract Background Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. Results One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues. Conclusions A collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins. PMID:22047182

Methods for high yield production of terpenes

DOEpatents

Kutchan, Toni; Higashi, Yasuhiro; Feng, Xiaohong

2017-01-03

Provided are enhanced high yield production systems for producing terpenes in plants via the expression of fusion proteins comprising various combinations of geranyl diphosphate synthase large and small subunits and limonene synthases. Also provided are engineered oilseed plants that accumulate monoterpene and sesquiterpene hydrocarbons in their seeds, as well as methods for producing such plants, providing a system for rapidly engineering oilseed crop production platforms for terpene-based biofuels.

Utility of high-resolution accurate MS to eliminate interferences in the bioanalysis of ribavirin and its phosphate metabolites.

PubMed

Wei, Cong; Grace, James E; Zvyaga, Tatyana A; Drexler, Dieter M

2012-08-01

The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5´-triphosphate (RTP), and also possibly through ribavirin 5´-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5´-diphosphate and RMP). In a recombinant human UGT1A1 assay, the assay buffer components uridine and its phosphorylated derivatives are isobaric with RBV and its phosphorylated metabolites, leading to significant interference when analyzed by LC-MS with the nominal mass resolution mode. Presented here is a LC-MS method employing LC coupled with full-scan high-resolution accurate MS analysis for the simultaneous quantitative determination of RBV, RMP, ribavirin 5´-diphosphate and RTP by differentiating RBV and its phosphorylated metabolites from uridine and its phosphorylated derivatives by accurate mass, thus avoiding interference. The developed LC-high-resolution accurate MS method allows for quantitation of RBV and its phosphorylated metabolites, eliminating the interferences from uridine and its phosphorylated derivatives in recombinant human UGT1A1 assays.

The stringent response regulates adaptation to darkness in the cyanobacterium Synechococcus elongatus.

PubMed

Hood, Rachel D; Higgins, Sean A; Flamholz, Avi; Nichols, Robert J; Savage, David F

2016-08-16

The cyanobacterium Synechococcus elongatus relies upon photosynthesis to drive metabolism and growth. During darkness, Synechococcus stops growing, derives energy from its glycogen stores, and greatly decreases rates of macromolecular synthesis via unknown mechanisms. Here, we show that the stringent response, a stress response pathway whose genes are conserved across bacteria and plant plastids, contributes to this dark adaptation. Levels of the stringent response alarmone guanosine 3'-diphosphate 5'-diphosphate (ppGpp) rise after a shift from light to dark, indicating that darkness triggers the same response in cyanobacteria as starvation in heterotrophic bacteria. High levels of ppGpp are sufficient to stop growth and dramatically alter many aspects of cellular physiology, including levels of photosynthetic pigments and polyphosphate, DNA content, and the rate of translation. Cells unable to synthesize ppGpp display pronounced growth defects after exposure to darkness. The stringent response regulates expression of a number of genes in Synechococcus, including ribosomal hibernation promoting factor (hpf), which causes ribosomes to dimerize in the dark and may contribute to decreased translation. Although the metabolism of Synechococcus differentiates it from other model bacterial systems, the logic of the stringent response remains remarkably conserved, while at the same time having adapted to the unique stresses of the photosynthetic lifestyle.

Monoterpene biosynthesis potential of plant subcellular compartments.

PubMed

Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

2016-01-01

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

The stringent response regulates adaptation to darkness in the cyanobacterium Synechococcus elongatus

PubMed Central

Hood, Rachel D.; Higgins, Sean A.; Flamholz, Avi; Nichols, Robert J.

2016-01-01

The cyanobacterium Synechococcus elongatus relies upon photosynthesis to drive metabolism and growth. During darkness, Synechococcus stops growing, derives energy from its glycogen stores, and greatly decreases rates of macromolecular synthesis via unknown mechanisms. Here, we show that the stringent response, a stress response pathway whose genes are conserved across bacteria and plant plastids, contributes to this dark adaptation. Levels of the stringent response alarmone guanosine 3′-diphosphate 5′-diphosphate (ppGpp) rise after a shift from light to dark, indicating that darkness triggers the same response in cyanobacteria as starvation in heterotrophic bacteria. High levels of ppGpp are sufficient to stop growth and dramatically alter many aspects of cellular physiology, including levels of photosynthetic pigments and polyphosphate, DNA content, and the rate of translation. Cells unable to synthesize ppGpp display pronounced growth defects after exposure to darkness. The stringent response regulates expression of a number of genes in Synechococcus, including ribosomal hibernation promoting factor (hpf), which causes ribosomes to dimerize in the dark and may contribute to decreased translation. Although the metabolism of Synechococcus differentiates it from other model bacterial systems, the logic of the stringent response remains remarkably conserved, while at the same time having adapted to the unique stresses of the photosynthetic lifestyle. PMID:27486247

Tomato linalool synthase is induced in trichomes by jasmonic acid

PubMed Central

van Schie, Chris C. N.; Haring, Michel A.

2007-01-01

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato. PMID:17440821

Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

PubMed Central

Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

2014-01-01

Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

Metabolic engineering of higher plants and algae for isoprenoid production.

PubMed

Kempinski, Chase; Jiang, Zuodong; Bell, Stephen; Chappell, Joe

2015-01-01

Isoprenoids are a class of compounds derived from the five carbon precursors, dimethylallyl diphosphate, and isopentenyl diphosphate. These molecules present incredible natural chemical diversity, which can be valuable for humans in many aspects such as cosmetics, agriculture, and medicine. However, many terpenoids are only produced in small quantities by their natural hosts and can be difficult to generate synthetically. Therefore, much interest and effort has been directed toward capturing the genetic blueprint for their biochemistry and engineering it into alternative hosts such as plants and algae. These autotrophic organisms are attractive when compared to traditional microbial platforms because of their ability to utilize atmospheric CO2 as a carbon substrate instead of supplied carbon sources like glucose. This chapter will summarize important techniques and strategies for engineering the accumulation of isoprenoid metabolites into higher plants and algae by choosing the correct host, avoiding endogenous regulatory mechanisms, and optimizing potential flux into the target compound. Future endeavors will build on these efforts by fine-tuning product accumulation levels via the vast amount of available "-omic" data and devising metabolic engineering schemes that integrate this into a whole-organism approach. With the development of high-throughput transformation protocols and synthetic biology molecular tools, we have only begun to harness the power and utility of plant and algae metabolic engineering.

Intensification of ion exchange desorption of thiamine diphosphate by low-powered ultrasound.

PubMed

Pinchukova, Natalia A; Voloshko, Alexander Y; Merko, Maria A; Bondarenko, Yana A; Chebanov, Valentin A

2018-03-01

The process of ultrasound-assisted ion-exchange desorption of cocarboxylase (thiamine diphosphate (TDP)) from a strong acidic cation resin was studied. Kinetics studies revealed that ultrasound accelerates TDP desorption by 3 times. The optimal desorption parameters, viz. US power input, sonication time, eluent/resin ratio and the eluent (ammonium acetate buffer) concentration were established which were 15mW/cm 3 , 20min, 1:1 and 1M, respectively. The resin stability studies showed that the optimal ultrasonic power was less by the order than the resin degradation threshold which ensures durable and efficient resin exploitation during production. The resin sorption capacity remained unchanged even after 20 cycles of TDP sorption, ultrasonic desorption and resin regeneration. The recovery ratio of TDP was shown to increase non-linearly with decreasing the resin saturation factor, which can be attributed to diffusion limitations occurring during desorption. The optimal resin loading corresponding to more than 90 per cent of TDP recovery was found to be at the level of 10 per cent of the maximal sorption capacity. The study revealed 4-5-fold increase in concentrations of the recovered solutions, which together with process times shortening should result in considerable energy saving in downstream operations on production scale. Copyright © 2017 Elsevier B.V. All rights reserved.

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

PubMed

Kasparyan, Elena; Richter, Michael; Dresen, Carola; Walter, Lydia S; Fuchs, Georg; Leeper, Finian J; Wacker, Tobias; Andrade, Susana L A; Kolter, Geraldine; Pohl, Martina; Müller, Michael

2014-12-01

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

Optimizing Associative Experimental Design for Protein Crystallization Screening

PubMed Central

Dinç, Imren; Pusey, Marc L.; Aygün, Ramazan S.

2016-01-01

The goal of protein crystallization screening is the determination of the main factors of importance to crystallizing the protein under investigation. One of the major issues about determining these factors is that screening is often expanded to many hundreds or thousands of conditions to maximize combinatorial chemical space coverage for maximizing the chances of a successful (crystalline) outcome. In this paper, we propose an experimental design method called “Associative Experimental Design (AED)” and an optimization method includes eliminating prohibited combinations and prioritizing reagents based on AED analysis of results from protein crystallization experiments. AED generates candidate cocktails based on these initial screening results. These results are analyzed to determine those screening factors in chemical space that are most likely to lead to higher scoring outcomes, crystals. We have tested AED on three proteins derived from the hyperthermophile Thermococcus thioreducens, and we applied an optimization method to these proteins. Our AED method generated novel cocktails (count provided in parentheses) leading to crystals for three proteins as follows: Nucleoside diphosphate kinase (4), HAD superfamily hydrolase (2), Nucleoside kinase (1). After getting promising results, we have tested our optimization method on four different proteins. The AED method with optimization yielded 4, 3, and 20 crystalline conditions for holo Human Transferrin, archaeal exosome protein, and Nucleoside diphosphate kinase, respectively. PMID:26955046

Chemical test for mammalian feces in grain products: collaborative study.

PubMed

Gerber, H R

1989-01-01

A collaborative study was conducted to validate the use of the AOAC alkaline phosphatase method for mammalian feces in corn meal, 44.B01-44.B06, for 7 additional products: brown rice cream, oat bran, grits, semolina, pasta flour, farina, and barley plus (a mixture of barley, oat bran, and brown rice). The proposed method determines the presence of alkaline phosphatase, an enzyme contained in mammalian feces, by using phenolphthalein diphosphate as the enzyme substrate in a test agar medium. Fecal matter is separated from the grain products by specific gravity differences in 1% test agar. As the product is distributed on liquid test agar, fecal fragments float while the grain products sink. The alkaline phosphatase cleaves phosphate radicals from phenolphthalein diphosphate, generating free phenolphthalein, which produces a pink to red-purple color around the fecal particles in the previously colorless medium. Collaborators' recovery averages ranged from 21.7 particles (72.3%) for oat bran to 25.3 particles (84.3%) for semolina at the 30 particle spike level. Overall average background was 0.4 positive reactions per food type. The collaborators reported that the method was quick, simple, and easy to use. The method has been approved interim official first action for all 7 grain products.

Purinergic signaling pathways in endocrine system.

PubMed

Bjelobaba, Ivana; Janjic, Marija M; Stojilkovic, Stanko S

2015-09-01

Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. Published by Elsevier B.V.

Purinergic Signaling Pathways in Endocrine System

PubMed Central

Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

2015-01-01

Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. PMID:25960051

Cloning and Functional Characterization of a β-Pinene Synthase from Artemisia annua That Shows a Circadian Pattern of Expression1

PubMed Central

Lu, Shan; Xu, Ran; Jia, Jun-Wei; Pang, Jihai; Matsuda, Seiichi P.T.; Chen, Xiao-Ya

2002-01-01

Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (−)-β-pinene and (−)-α-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (−)-β-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern. PMID:12226526

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis1[W

PubMed Central

Ogawa, Takahisa; Yoshimura, Kazuya; Miyake, Hiroe; Ishikawa, Kazuya; Ito, Daisuke; Tanabe, Noriaki; Shigeoka, Shigeru

2008-01-01

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its Km value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 μm) than for ADP-glucose (1,235 ± 65 μm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 μm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 μm) and NADPH (36.9 ± 3.5 μm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 μm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes. PMID:18815383

Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection

PubMed Central

Gao, Wei; Chua, Kyra; Davies, John K.; Newton, Hayley J.; Seemann, Torsten; Harrison, Paul F.; Holmes, Natasha E.; Rhee, Hyun-Woo; Hong, Jong-In; Hartland, Elizabeth L.; Stinear, Timothy P.; Howden, Benjamin P.

2010-01-01

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens. PMID:20548948

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata

PubMed Central

Hussain, Tajammul; Plunkett, Blue; Ejaz, Mahwish; Espley, Richard V.; Kayser, Oliver

2018-01-01

The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

Chlorophyll Degradation: The Tocopherol Biosynthesis-Related Phytol Hydrolase in Arabidopsis Seeds Is Still Missing1[C][W][OPEN

PubMed Central

Zhang, Wei; Liu, Tianqi; Ren, Guodong; Hörtensteiner, Stefan; Zhou, Yongming; Cahoon, Edgar B.; Zhang, Chunyu

2014-01-01

Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn’t enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis. PMID:25059706

Biochemical Characterization and Homology Modeling of Methylbutenol Synthase and Implications for Understanding Hemiterpene Synthase Evolution in Plants*

PubMed Central

Gray, Dennis W.; Breneman, Steven R.; Topper, Lauren A.; Sharkey, Thomas D.

2011-01-01

2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ∼90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K+, whereas isoprene production is inhibited by K+ such that, at physiologically relevant [K+], little or no isoprene emission should be detected from MBO-emitting trees. The Km of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site. PMID:21504898

Synthesis and Screening of New Antimalarial Drugs

DTIC Science & Technology

1987-10-30

correlate well with the known pIharmacokinetics of thie drug. 3. fhe blood schizonticidal properties of chloroquine (active at 3 mg/kg/day x 7 days) were...Reference drug chloroquine has shown consistently curative action at 3 mg/kg (base) x 7 days. No escalation of chloroquine curative dose has been...from patent infection has been used from time to time for standardization of blood schizontocidal test using chloroquine diphosphate as the reference

The effect of 24S-hydroxycholesterol on cholesterol homeostasis in neurons: quantitative changes to the cortical neuron proteome.

PubMed

Wang, Yuqin; Muneton, Sabina; Sjövall, Jan; Jovanovic, Jasmina N; Griffiths, William J

2008-04-01

In humans, the brain represents only about 2% of the body's mass but contains about one-quarter of the body's free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 2.3.3.10), diphosphomevalonate decarboxylase (EC 4.1.1.33), isopentenyl-diphosphate delta isomerase (EC 5.3.3.2), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 2.5.1.10), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 2.5.1.21) and methylsterol monooxygenase (EC 1.14.13.72). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not found to be down-regulated in neurons treated with 24S-hydroxycholesterol, while apolipoprotein E (apo E), a cholesterol trafficking protein, was found to be up-regulated. Taken together, this data leads to the hypothesis that, in times of cholesterol excess, 24S-hydroxycholesterols signals down-regulation of cholesterol synthesis enzymes through SREBP-2, but up-regulates apo E synthesis (through the liver X receptor) leading to cholesterol storage and restoration of cholesterol balance.

Next-generation sequencing (NGS) transcriptomes reveal association of multiple genes and pathways contributing to secondary metabolites accumulation in tuberous roots of Aconitum heterophyllum Wall.

PubMed

Pal, Tarun; Malhotra, Nikhil; Chanumolu, Sree Krishna; Chauhan, Rajinder Singh

2015-07-01

The transcriptomes of Aconitum heterophyllum were assembled and characterized for the first time to decipher molecular components contributing to biosynthesis and accumulation of metabolites in tuberous roots. Aconitum heterophyllum Wall., popularly known as Atis, is a high-value medicinal herb of North-Western Himalayas. No information exists as of today on genetic factors contributing to the biosynthesis of secondary metabolites accumulating in tuberous roots, thereby, limiting genetic interventions towards genetic improvement of A. heterophyllum. Illumina paired-end sequencing followed by de novo assembly yielded 75,548 transcripts for root transcriptome and 39,100 transcripts for shoot transcriptome with minimum length of 200 bp. Biological role analysis of root versus shoot transcriptomes assigned 27,596 and 16,604 root transcripts; 12,340 and 9398 shoot transcripts into gene ontology and clusters of orthologous group, respectively. KEGG pathway mapping assigned 37 and 31 transcripts onto starch-sucrose metabolism while 329 and 341 KEGG orthologies associated with transcripts were found to be involved in biosynthesis of various secondary metabolites for root and shoot transcriptomes, respectively. In silico expression profiling of the mevalonate/2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway genes for aconites biosynthesis revealed 4 genes HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), MVDD (mevalonate diphosphate decarboxylase) and HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase) with higher expression in root transcriptome compared to shoot transcriptome suggesting their key role in biosynthesis of aconite alkaloids. Five genes, GMPase (geranyl diphosphate mannose pyrophosphorylase), SHAGGY, RBX1 (RING-box protein 1), SRF receptor kinases and β-amylase, implicated in tuberous root formation in other plant species showed higher levels of expression in tuberous roots compared to shoots. A total of 15,487 transcription factors belonging to bHLH, MYB, bZIP families and 399 ABC transporters which regulate biosynthesis and accumulation of bioactive compounds were identified in root and shoot transcriptomes. The expression of 5 ABC transporters involved in tuberous root development was validated by quantitative PCR analysis. Network connectivity diagrams were drawn for starch-sucrose metabolism and isoquinoline alkaloid biosynthesis associated with tuberous root growth and secondary metabolism, respectively, in root transcriptome of A. heterophyllum. The current endeavor will be of practical importance in planning a suitable genetic intervention strategy for the improvement of A. heterophyllum.

Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex - Linkage of protein dynamics to catalysis

PubMed Central

Jordan, Frank; Arjunan, Palaniappa; Kale, Sachin; Nemeria, Natalia S.; Furey, William

2009-01-01

The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an 19F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex. PMID:20160956

Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

NASA Technical Reports Server (NTRS)

Rounds, M. A.; Nielsen, S. S.; Mitchell, C. A. (Principal Investigator)

1993-01-01

The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.

Characterization of a Dopaminergic Stimulatory Factor Derived from Monoclonal Cell Lines of Striatal Origin

DTIC Science & Technology

2006-12-01

other substrates for its biosynthesis would be effective in raising MN9D dopamine content. The pyrimidine, cytidine triphosphate (CTP) reacts with...phosphatidylethanolamine or phosphatidylcholine. MN9D cells were treated for 48 hrs with 2 mM CTP, cytidine diphosphate (CDP) or cytidine monophosphate (CMP), in the...presence or absence of 25 µM ethanolamine or 25 µM phosphoethanolamine. Cytidine alone did not alter the dopaminergic stimulatory effect of

Engineering yeasts as platform organisms for cannabinoid biosynthesis.

PubMed

Zirpel, Bastian; Degenhardt, Friederike; Martin, Chantale; Kayser, Oliver; Stehle, Felix

2017-10-10

Δ 9 -tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis satival. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme's coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells

PubMed Central

Guo, Jianru; Chen, QianQian; Lam, Christopher Wai Kei; Wang, Caiyun; Wong, Vincent Kam Wai; Xu, Fengguo; Jiang, ZhiHong; Zhang, Wei

2015-01-01

Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance. PMID:26578061

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria.

PubMed

Artsatbanov, V Yu; Vostroknutova, G N; Shleeva, M O; Goncharenko, A V; Zinin, A I; Ostrovsky, D N; Kapreliants, A S

2012-04-01

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) - an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) - most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82-92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes'', well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

PubMed

Sato, Akira; Takano, Takeshi; Hiramoto, Akiko; Naito, Tomoharu; Matsuda, Akira; Fukushima, Masakazu; Wataya, Yusuke; Kim, Hye-Sook

2017-08-01

A nucleosidic medicine, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III

PubMed Central

Karačić, Zrinka; Vukelić, Bojana; Ho, Gabrielle H.; Jozić, Iva; Sučec, Iva; Salopek-Sondi, Branka; Kozlović, Marija; Brenner, Steven E.; Ludwig-Müller, Jutta; Abramić, Marija

2017-01-01

In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyses the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase. PMID:27467751

Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group-specific relationships.

PubMed

Freas, Nicholas; Newton, Peter; Perozich, John

2016-01-01

UDP-glucose dehydrogenase (UDPGDH), UDP-N-acetyl-mannosamine dehydrogenase (UDPNAMDH) and GDP-mannose dehydrogenase (GDPMDH) belong to a family of NAD (+)-linked 4-electron-transfering oxidoreductases called nucleotide diphosphate sugar dehydrogenases (NDP-SDHs). UDPGDH is an enzyme responsible for converting UDP-d-glucose to UDP-d-glucuronic acid, a product that has different roles depending on the organism in which it is found. UDPNAMDH and GDPMDH convert UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid and GDP-mannose to GDP-mannuronic acid, respectively, by a similar mechanism to UDPGDH. Their products are used as essential building blocks for the exopolysaccharides found in organisms like Pseudomonas aeruginosa and Staphylococcus aureus. Few studies have investigated the relationships between these enzymes. This study reveals the relationships between the three enzymes by analysing 229 amino acid sequences. Eighteen invariant and several other highly conserved residues were identified, each serving critical roles in maintaining enzyme structure, coenzyme binding or catalytic function. Also, 10 conserved motifs that included most of the conserved residues were identified and their roles proposed. A phylogenetic tree demonstrated relationships between each group and verified group assignment. Finally, group entropy analysis identified novel conservations unique to each NDP-SDH group, including residue positions critical to NDP-sugar substrate interaction, enzyme structure and intersubunit contact. These positions may serve as targets for future research. UDP-glucose dehydrogenase (UDPGDH, EC 1.1.1.22).

Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity.

PubMed

Gibson, Kyle R; Neilson, Ilene L; Barrett, Fiona; Winterburn, Tim J; Sharma, Sushma; MacRury, Sandra M; Megson, Ian L

2009-10-01

N-Acetylcysteine (NAC) is a frequently used "antioxidant" in vitro, but the concentrations applied rarely correlate with those encountered with oral dosing in vivo. Here, we investigated the in vitro antioxidant and antiplatelet properties of NAC at concentrations (10-100 microM) that are achievable in plasma with tolerable oral dosing. The impact of NAC pretreatment (2 hours) on aggregation of platelets from healthy volunteers in response to thrombin and adenosine diphosphate and on platelet-derived nitric oxide (NO) was examined. NAC was found to be a weak reducing agent and a poor antioxidant compared with glutathione (reduced form) (GSH). However, platelets treated with NAC showed enhanced antioxidant activity and depression of reactive oxygen species generation associated with increases in intraplatelet GSH levels. An approximately 2-fold increase in NO synthase-derived nitrite was observed with 10 microM NAC treatment, but the effect was not concentration dependent. Finally, NAC significantly reduced both thrombin-induced and adenosine diphosphate-induced platelet aggregation. NAC should be considered a weak antioxidant that requires prior conversion to GSH to convey antioxidant and antithrombotic benefit at therapeutically relevant concentrations. Our results suggest that NAC might be an effective antiplatelet agent in conditions where increased oxidative stress contributes to heightened risk of thrombosis but only if the intraplatelet machinery to convert it to GSH is functional.

Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites.

PubMed

Vieira, Plínio Salmazo; Souza, Tatiana de Arruda Campos Brasil; Honorato, Rodrigo Vargas; Zanphorlin, Letícia Maria; Severiano, Kelven Ulisses; Rocco, Silvana Aparecida; de Oliveira, Arthur Henrique Cavalcante; Cordeiro, Artur Torres; Oliveira, Paulo Sérgio Lopes; de Giuseppe, Priscila Oliveira; Murakami, Mário Tyago

2017-07-01

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Guanosine 5‧-diphosphate as Potential Iron Mobilizer: Preventing the Hepcidin-Ferroportin Interaction and Modulating the Interleukin-6/Stat-3 Pathway

NASA Astrophysics Data System (ADS)

Angmo, Stanzin; Tripathi, Neha; Abbat, Sheenu; Sharma, Shailesh; Singh, Shelley Sardul; Halder, Avishek; Yadav, Kamalendra; Shukla, Geeta; Sandhir, Rajat; Rishi, Vikas; Bharatam, Prasad V.; Yadav, Hariom; Singhal, Nitin Kumar

2017-01-01

Hepcidin, a peptide hormone, is a key regulator in mammalian iron homeostasis. Increased level of hepcidin due to inflammatory conditions stimulates the ferroportin (FPN) transporter internalization, impairing the iron absorption; clinically manifested as anemia of inflammation (AI). Inhibiting hepcidin-mediated FPN degradation is proposed as an important strategy to combat AI. A systematic approach involving in silico, in vitro, ex vivo and in vivo studies is employed to identify hepcidin-binding agents. The virtual screening of 68,752 natural compounds via molecular docking resulted into identification of guanosine 5‧-diphosphate (GDP) as a promising hepcidin-binding agent. The molecular dynamics simulations helped to identify the important hepcidin residues involved in stabilization of hepcidin-GDP complex. The results gave a preliminary indication that GDP may possibly inhibit the hepcidin-FPN interactions. The in vitro studies revealed that GDP caused FPN stabilization (FPN-GFP cell lines) and increased the FPN-mediated cellular iron efflux (HepG2 and Caco-2 cells). Interestingly, the co-administration of GDP and ferrous sulphate (FeSO4) ameliorated the turpentine-induced AI in mice (indicated by increased haemoglobin level, serum iron, FPN expression and decreased ferritin level). These results suggest that GDP a promising natural small-molecule inhibitor that targets Hepcidin-FPN complex may be incorporated with iron supplement regimens to ameliorate AI.

Intracellular Distribution of Enzymes of the Cytidine Diphosphate Choline Pathway in Castor Bean Endosperm

PubMed Central

Lord, J. M.; Kagawa, T.; Beevers, Harry

1972-01-01

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 × g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 × g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm3 contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm2. Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm3, and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm3. Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components. Images PMID:4506764

Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 degrees C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armstrong, Christopher R.; Felmy, Andrew R.; Clark, Sue B.

2010-11-01

In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO2)(3)(PO4)(2)center dot 4H(2)O) and Na autunite (Na[UO2PO4]center dot xH(2)O) at 23 and 50 degrees C in NaClO4-HClO4 solutions at pC(H+) = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 in solutions were equilibrated at 23 and 50 degrees C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditionsmore » were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K-sp, for TDT was determined to be -49.7 and -51.3 at 23 and 50 degrees C respectively. log K for Na autunite was determined to be -24.4 (23 degrees C) and -24.1 +/- 0.2 (50 degrees C).« less

Functional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera.

PubMed

Abbassi, Shakeel; Patel, Krunal; Khan, Bashir; Bhosale, Siddharth; Gaikwad, Sushama

2016-02-01

Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40°C and transient exposure of hydrophobic residues at 50°C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher KSV for KI as compared to that for CsCl. The native enzyme displayed two different lifetimes, τ1 (1.203±0.036 ns) and τ2 (3.473±0.12 ns) indicating two populations of tryptophan. Copyright © 2015 Elsevier B.V. All rights reserved.

Sterol composition and biosynthetic genes of the recently discovered photosynthetic alveolate, Chromera velia (chromerida), a close relative of apicomplexans.

PubMed

Leblond, Jeffrey D; Dodson, Joshua; Khadka, Manoj; Holder, Sabrina; Seipelt, Rebecca L

2012-01-01

Chromera velia is a recently discovered, photosynthetic, marine alveolate closely related to apicomplexan parasites, and more distantly to perkinsids and dinoflagellates. To date, there are no published studies on the sterols of C. velia. Because apicomplexans and perkinsids are not known to synthesize sterols de novo, but rather obtain them from their host organisms, our objective was to examine the composition of the sterols of C. velia to assess whether or not there is any commonality with dinoflagellates as the closest taxonomic group capable of synthesizing sterols de novo. Furthermore, knowledge of the sterols of C. velia may provide insight into the sterol biosynthetic capabilities of apicomplexans prior to loss of sterol biosynthesis. We have found that C. velia possesses two primary sterols, 24-ethylcholesta-5,22E-dien-3β-ol, and 24-ethylcholest-5-en-3β-ol, not common to dinoflagellates, but rather commonly found in other classes of algae and plants. In addition, we have identified computationally three genes, SMT1 (sterol-24C-methyltransferase), FDFT1 (farnesyl diphosphate farnesyl transferase, squalene synthase), and IDI1 (isopentenyl diphosphate Δ-isomerase), predicted to be involved in sterol biosynthesis by their similarity to analogous genes in other sterol-producing eukaryotes, including a number of algae. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

PubMed Central

Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

1983-01-01

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

The Effect of Nucleotides and Inhibitors on Respiration in Isolated Wheat Mitochondria 1

PubMed Central

Pomeroy, M. Keith

1975-01-01

The effect of mono-, di-, and trinucleoside phosphates and respiratory inhibitors on respiration in winter wheat (Triticum aestivum L. cv. Rideau) mitochondria has been examined. When added during state 4 respiration, subsequent to addition of ADP, all of the dinucleotides stimulated oxidation and induced respiratory control with all substrates examined. Similar results were obtained with AMP, but other mononucleotides and all trinucleotides did not affect the rate of oxidation. Nucleoside diphosphates did not stimulate respiration when added prior to the addition of ADP, but subsequent addition of AMP, ADP, or ATP re-established coupled respiration in the presence of the dinucleotides. The duration of 2, 4-dinitrophenol stimulated respiration during oxidation of α-ketoglutarate was found to be dependent on the amount of AMP, ADP, or ATP added, either prior, or subsequent to, addition of the uncoupler. The addition of oligomycin during 2, 4-dinitrophenol stimulated respiration reestablished coupled respiration with low ADP/O ratios, when added after addition of ATP or conditions which allow formation of ATP from added ADP. The nucleoside diphosphates, other than ADP, did not stimulate oxidation of α-ketoglutarate in the presence of 2, 4-dinitrophenol until a small amount of adenine nucleotide was added to the system. The results suggest that dinucleotides other than ADP, are able to participate in the energy conversion processs of the mitochondria, probably via transphosphorylation reactions. Images PMID:16659027

Actions of p-synephrine on hepatic enzyme activities linked to carbohydrate metabolism and ATP levels in vivo and in the perfused rat liver.

PubMed

Maldonado, Marcos Rodrigues; Bracht, Lívia; de Sá-Nakanishi, Anacharis Babeto; Corrêa, Rúbia Carvalho Gomes; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

2018-01-01

p-Synephrine is one of the main active components of the fruit of Citrus aurantium (bitter orange). Extracts of the bitter orange and other preparations containing p-synephrine have been used worldwide to promote weight loss and for sports performance. The purpose of the study was to measure the action of p-synephrine on hepatic enzyme activities linked to carbohydrate and energy metabolism and the levels of adenine mononucleotides. Enzymes and adenine mononucleotides were measured in the isolated perfused rat liver and in vivo after oral administration of the drug (50 and 300 mg/kg) by using standard techniques. p-Synephrine increased the activity of glycogen phosphorylase in vivo and in the perfused liver. It decreased, however, the activities of pyruvate kinase and pyruvate dehydrogenase also in vivo and in the perfused liver. p-Synephrine increased the hepatic pools of adenosine diphosphate and adenosine triphosphate. Stimulation of glycogen phosphorylase is consistent with the reported increased glycogenolysis in the perfused liver and increased glycemia in rats. The decrease in the pyruvate dehydrogenase activity indicates that p-synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. The capability of increasing the adenosine triphosphate-adenosine diphosphate pool indicates a beneficial effect of p-synephrine on the cellular energetics. Copyright © 2017 John Wiley & Sons, Ltd.

Solanesyl Diphosphate Synthase, an Enzyme of the Ubiquinone Synthetic Pathway, Is Required throughout the Life Cycle of Trypanosoma brucei

PubMed Central

Lai, De-Hua; Poropat, Estefanía; Pravia, Carlos; Landoni, Malena; Couto, Alicia S.; Pérez Rojo, Fernando G.; Fuchs, Alicia G.; Dubin, Marta; Elingold, Igal; Rodríguez, Juan B.; Ferella, Marcela; Esteva, Mónica I.

2014-01-01

Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target. PMID:24376001

Chemistry and biotechnology of carotenoids.

PubMed

Namitha, K K; Negi, P S

2010-09-01

Carotenoids are one of the most widespread groups of pigments in nature and more than 600 of these have been identified. Beside provitamin A activity, carotenoids are important as antioxidants and protective agents against various diseases. They are isoprenoids with a long polyene chain containing 3 to 15 conjugated double bonds, which determines their absorption spectrum. Cyclization at one or both ends occurs in hydrocarbon carotene, while xanthophylls are formed by the introduction of oxygen. In addition, modifications involving chain elongation, isomerization, or degradation are also found. The composition of carotenoids in food may vary depending upon production practices, post-harvest handling, processing, and storage. In higher plants they are synthesized in the plastid. Both mevalonate dependent and independent pathway for the formation of isopentenyl diphosphate are known. Isopentenyl diphosphate undergoes a series of addition and condensation reactions to form phytoene, which gets converted to lycopene. Cyclization of lycopene either leads to the formation of β-carotene and its derivative xanthophylls, β-cryptoxanthin, zeaxanthin, antheraxanthin, and violaxanthin or α-carotene and lutein. Even though most of the carotenoid biosynthetic genes have been cloned and identified, some aspects of carotenoid formation and manipulation in higher plants especially remain poorly understood. In order to enhance the carotenoid content of crop plants to a level that will be required for the prevention of diseases, there is a need for research in both the basic and the applied aspects.

Influence of uridine diphosphate glucuronosyltransferase 2B7 -161C>T polymorphism on the concentration of valproic acid in pediatric epilepsy patients.

PubMed

Inoue, Kazuyuki; Suzuki, Eri; Yazawa, Rei; Yamamoto, Yoshiaki; Takahashi, Toshiki; Takahashi, Yukitoshi; Imai, Katsumi; Koyama, Seiichi; Inoue, Yushi; Tsuji, Daiki; Hayashi, Hideki; Itoh, Kunihiko

2014-06-01

Valproic acid (VPA) is widely used to treat various types of epilepsy. Interindividual variability in VPA pharmacokinetics may arise from genetic polymorphisms of VPA-metabolizing enzymes. This study aimed to examine the relationships between plasma VPA concentrations and the -161C>T single nucleotide polymorphism in uridine diphosphate glucuronosyltransferase (UGT) 2B7 genes in pediatric epilepsy patients. This study included 78 pediatric epilepsy patients carrying the cytochrome P450 (CYP) 2C9*1/*1 genotype and who were not treated with the enzyme inducers (phenytoin, phenobarbital, and carbamazepine), lamotrigine, and/or topiramate. CYP2C9*3 and UGT2B7 -161C>T polymorphisms were identified using methods based on polymerase chain reaction-restriction fragment length polymorphism. Blood samples were drawn from each patient under steady-state conditions, and plasma VPA concentrations were measured. Significant differences in adjusted plasma VPA concentrations were observed between carriers of CC, CT, and TT genotypes in the UGT2B7 -161C>T polymorphism (P = 0.039). Patients with the CC genotype had lower adjusted plasma VPA concentrations than those with CT or TT genotype (P = 0.028). These data suggest that the UGT2B7 -161C>T polymorphism in pediatric epilepsy patients carrying the CYP2C9*1/*1 genotype affects VPA concentration.

Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo.

PubMed

Bapiro, T E; Frese, K K; Courtin, A; Bramhall, J L; Madhu, B; Cook, N; Neesse, A; Griffiths, J R; Tuveson, D A; Jodrell, D I; Richards, F M

2014-07-15

The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS(G12D); p53(R172H); pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism. LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay. In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition. GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.

Characterization of Leachianone G 2″ -Dimethylallyltransferase, a Novel Prenyl Side-Chain Elongation Enzyme for the Formation of the Lavandulyl Group of Sophoraflavanone G in Sophora flavescens Ait. Cell Suspension Cultures

PubMed Central

Zhao, Ping; Inoue, Kenichiro; Kouno, Isao; Yamamoto, Hirobumi

2003-01-01

Leachianone G (LG) 2′′-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2′′ position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2′-hydroxy function in LG was indispensable to the activity. The apparent Km values for dimethylallyl diphosphate and LG were 59 and 2.3 μm, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2′′-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2′-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups. PMID:14551337

Identification of Guanosine 5′-diphosphate as Potential Iron Mobilizer: Preventing the Hepcidin-Ferroportin Interaction and Modulating the Interleukin-6/Stat-3 Pathway

PubMed Central

Angmo, Stanzin; Tripathi, Neha; Abbat, Sheenu; Sharma, Shailesh; Singh, Shelley Sardul; Halder, Avishek; Yadav, Kamalendra; Shukla, Geeta; Sandhir, Rajat; Rishi, Vikas; Bharatam, Prasad V.; Yadav, Hariom; Singhal, Nitin Kumar

2017-01-01

Hepcidin, a peptide hormone, is a key regulator in mammalian iron homeostasis. Increased level of hepcidin due to inflammatory conditions stimulates the ferroportin (FPN) transporter internalization, impairing the iron absorption; clinically manifested as anemia of inflammation (AI). Inhibiting hepcidin-mediated FPN degradation is proposed as an important strategy to combat AI. A systematic approach involving in silico, in vitro, ex vivo and in vivo studies is employed to identify hepcidin-binding agents. The virtual screening of 68,752 natural compounds via molecular docking resulted into identification of guanosine 5′-diphosphate (GDP) as a promising hepcidin-binding agent. The molecular dynamics simulations helped to identify the important hepcidin residues involved in stabilization of hepcidin-GDP complex. The results gave a preliminary indication that GDP may possibly inhibit the hepcidin-FPN interactions. The in vitro studies revealed that GDP caused FPN stabilization (FPN-GFP cell lines) and increased the FPN-mediated cellular iron efflux (HepG2 and Caco-2 cells). Interestingly, the co-administration of GDP and ferrous sulphate (FeSO4) ameliorated the turpentine-induced AI in mice (indicated by increased haemoglobin level, serum iron, FPN expression and decreased ferritin level). These results suggest that GDP a promising natural small-molecule inhibitor that targets Hepcidin-FPN complex may be incorporated with iron supplement regimens to ameliorate AI. PMID:28054602

Biphasic Elimination of Tenofovir Diphosphate and Nonlinear Pharmacokinetics of Zidovudine Triphosphate in a Microdosing Study

PubMed Central

Chen, Jianmeng; Flexner, Charles; Liberman, Rosa G.; Skipper, Paul L.; Louissaint, Nicolette; Tannenbaum, Steven R.; Hendrix, Craig; Fuchs, Edward

2012-01-01

Objective Phase 0 studies can provide initial pharmacokinetics (PK) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of two antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. Study Design We administered a microdose (100 μg) of 14C-labeled drug (ZDV or tenofovir disoproxil fumarate (TDF)) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in PBMCs and CD4+ cells were measured by AMS. Results The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 μg to 300 mg), while the intracellular TFV-DP PK were linear over the same dose range. ZDV-TP concentrations were lower in CD4+ cells versus total peripheral blood mononuclear cells (PBMCs), while TFV-DP concentrations were not different in CD4+ cells and PBMCs. Conclusion Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. AMS shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs. PMID:23187888

Synthesis of conformationally locked L-deoxythreosyl phosphonate nucleosides built on a bicyclo[3.1.0]hexane template.

PubMed

Saneyoshi, Hisao; Deschamps, Jeffrey R; Marquez, Victor E

2010-11-19

Two conformationally locked versions of l-deoxythreosyl phosphonate nucleosides (2 and 3) were synthesized to investigate the preference of HIV reverse transcriptase for a conformation displaying either a fully diaxial or fully diequatorial disposition of substituents. Synthesis of the enantiomeric 4-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-2-ol carbocyclic nucleoside precursors (diaxially disposed) proceeded straightforwardly from commercially available (1R,4S)-4-hydroxy-2-cyclopent-2-enyl-1-yl acetate employing a hydroxyl-directed Simmons-Smith cyclopropanation that culminated with a Mitsunobu coupling of the purine base. For the more complicated 1-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-3-ol carbocyclic nucleoside precursors (diequatorially disposed), the obligatory linear approach required the syntheses of key 1-aminobicyclo[3.1.0.]hexan-3-yl benzoate precursors that were assembled via the amide variant of the Kulinkovich reaction involving the intramolecular cyclopropanation of a substituted δ-vinylamide. Completion of the purine ring was achieved by conventional approaches but with much improved yields through the use of a microwave reactor. The syntheses of the phosphonates and the corresponding diphosphates were achieved by conventional means. None of the diphosphates, which were supposed to act as nucleoside triphosphate mimics, could compete with dATP even when present in a 10-fold excess.

Laticifer-specific cis-prenyltransferase silencing affects the rubber, triterpene, and inulin content of Taraxacum brevicorniculatum.

PubMed

Post, Janina; van Deenen, Nicole; Fricke, Julia; Kowalski, Natalie; Wurbs, David; Schaller, Hubert; Eisenreich, Wolfgang; Huber, Claudia; Twyman, Richard M; Prüfer, Dirk; Gronover, Christian Schulze

2012-03-01

Certain Taraxacum species, such as Taraxacum koksaghyz and Taraxacum brevicorniculatum, produce large amounts of high-quality natural rubber in their latex, the milky cytoplasm of specialized cells known as laticifers. This high-molecular mass biopolymer consists mainly of poly(cis-1,4-isoprene) and is deposited in rubber particles by particle-bound enzymes that carry out the stereospecific condensation of isopentenyl diphosphate units. The polymer configuration suggests that the chain-elongating enzyme (rubber transferase; EC 2.5.1.20) is a cis-prenyltransferase (CPT). Here, we present a comprehensive analysis of transgenic T. brevicorniculatum plants in which the expression of three recently isolated CPTs known to be associated with rubber particles (TbCPT1 to -3) was heavily depleted by laticifer-specific RNA interference (RNAi). Analysis of the CPT-RNAi plants by nuclear magnetic resonance, size-exclusion chromatography, and gas chromatography-mass spectrometry indicated a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and the main storage carbohydrate, inulin. Transmission electron microscopy revealed that the laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. We also observed lower activity of hydroxymethylglutaryl-coenzyme A reductase, the key enzyme in the mevalonate pathway, reflecting homeostatic control of the isopentenyl diphosphate pool. To our knowledge, this is the first in planta demonstration of latex-specific CPT activity in rubber biosynthesis.

Morphologic examination of CD3-CD4(bright) cells in rat liver.

PubMed

Yamamoto, Satoshi; Sato, Yosinobu; Abo, Toru; Hatakeyama, Katsuyosi

2002-01-01

Recently, we found CD3-CD4(bright) cells with comparative specificity for normal rat liver. In the current study, we investigated the type and form of both CD3-CD4(bright) cells and CD3-CD4(dull) cells in the rat liver. The surface phenotype of hepatic mononuclear cells in Lewis rats was identified by using monoclonal antibodies including anti-CD4, anti-CD3, and antimacrophage in conjunction with two- or three-color immunofluorescence analysis. CD3-CD4(bright) cells and CD3-CD4(dull) cells were examined morphologically using May-Giemsa staining and scanning electron microscopy. The distribution of CD3-CD4(bright) cells and CD3-CD4(dull) cells 48 hours after intravenous administration of liposome-encapsulated dichloromethylene diphosphate was also investigated. In comparison to CD3-CD4(dull) cells, CD3-CD4(bright) cells were slightly larger macrophages with abundant cytoplasmic granules, being present with comparative specificity for normal rat liver and showing negligible effects by intravenous liposome-encapsulated dichloromethylene diphosphate administration. These data suggest that in normal young rat liver these CD3-CD4(dull) and CD3-CD4(bright) cells may be dendritic cells and Kupffer cells that shift from the liver to the spleen or vice versa. These cells may also be able to locally proliferate in liver or spleen due to changes in the developing liver.

The characterization of ficaprenol-10, -11 and -12 from the leaves of Ficus elastica (decorative rubber plant)

PubMed Central

Stone, K. J.; Wellburn, A. R.; Hemming, F. W.; Pennock, J. F.

1967-01-01

Evidence from mass, nuclear-magnetic-resonance and infrared spectrometry and from gas–liquid and thin-layer chromatography is presented in favour of the presence of cis–trans-decaprenol, -undecaprenol and -dodecaprenol in the mixture of polyprenols (2·6mg./g.) isolated from leaf tissue of Ficus elastica. The trivial names ficaprenol-10, -11 and -12 are proposed. Nuclear-magnetic-resonance studies showed that each of these prenols contains three trans internal isoprene residues and a cis `OH-terminal' isoprene residue. Ficaprenol-11 is the major component of the mixture. Chromatographic evidence suggests the presence also of small amounts of ficaprenol-9 and -13. The precise position of the three trans internal isoprene residues was not determined but it is suggested that these are adjacent to the ω-terminal isoprene residue and that the ficaprenols are formed from all-trans-geranylgeranyl pyrophosphate. It is also suggested that ficaprenol-10, -11, -12 and -13 are probably the same compounds as castaprenol-10, -11, -12 and -13. PMID:6030292

Human Isoprenoid Synthase Enzymes as Therapeutic Targets

NASA Astrophysics Data System (ADS)

Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

2014-07-01

The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

Human isoprenoid synthase enzymes as therapeutic targets

PubMed Central

Park, Jaeok; Matralis, Alexios N.; Berghuis, Albert M.; Tsantrizos, Youla S.

2014-01-01

In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies. PMID:25101260

Carbon Dioxide Fixation in Isolated Kalanchoe Chloroplasts 1

PubMed Central

Levi, Carolyn; Gibbs, Martin

1975-01-01

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO2 per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO2 fixation were primarily those of the photosynthetic carbon reduction cycle. PMID:16659249

Malignant external otitis: The diagnostic value of bone scintigraphy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostfeld, E.; Aviel, A.; Pelet, D.

1981-06-01

Technetium99m Methylene Diphosphate bone scintigraphy (BS) of the skull was performed in three patients with malignant external otitis (MEO). Pathological uptake of the radioisotope in the mastoid region was found during the early stages of MEO updating radiologic findings. The extent of the radioisotope accumulation during the early stages of MEO indicates that the actual tissue damage exceeds the clinical estimation. The follow-up BS findings correlate well with the clinical course of MEO indicating either healing or extension to the base of skull.

Properties of lubrol-extracted uridine diphosphate glucuronyltransferase.

PubMed

Howland, R D; Burkhalter, A; Trevor, A J; Hegeman, S; Shirachi, D Y

1971-12-01

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.

Properties of Lubrol-extracted uridine diphosphate glucuronyltransferase

PubMed Central

Howland, R. D.; Burkhalter, A.; Trevor, A. J.; Hegeman, S.; Shirachi, D. Y.

1971-01-01

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction. PMID:5144269

Formation of nucleoside 5'-polyphosphates under potentially prebiological conditions

NASA Technical Reports Server (NTRS)

Lohrmann, R.

1976-01-01

The characteristics and efficiencies of biochemical reactions involving nucleoside 5'-diphosphates and -triphosphates (important substrates of RNA and DNA synthesis) under conditions corresponding to the primitive prebiotic earth are investigated. Urea catalysis of the formation of linear inorganic polyphosphates and metal ions promoting the reactions are discussed. Linear polyphosphate was incubated with Mg(++) in the presence of a nucleoside 5'-phosphate, to yield nucleoside 5'-polyphosphates when products are dried, while Mg(++) prompts depolymerization to trimetaphosphate in aqueous solutions. Plausible biogenetic pathways are examined.

Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

PubMed

Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-06-07

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

Regulation of PRPP and nucleoside tri and tetraphosphate pools in Escherichia coli under conditions of nitrogen starvation.

PubMed Central

Villadsen, I S; Michelsen, O

1977-01-01

The ribonucleoside triphosphate, deoxyribonucleoside triphosphate, 3' -diphosphate guanosine 5' -diphosphate (ppGpp), and 5-phosphoribosyl 1-pyrophosphate (PRPP) pools in Escherichia coli B were determined by thin-layer chromatography during changing conditions to ammonium starvation. The intracellular concentrations of all nucleotides were found to change in a well-defined order several minutes before andy observed change in the optical density of the culture. The levels of purine nucleoside triphosphates (adenosine 5' -triphosphate [CTP], dCTP) and uridine nucleotides (uridine 5' -triphosphate, deoxythymidine 5'-triphosphate). The deoxyribonucleotides thus behaved as the ribonucleotides. The levels of ppGpp increased 11-fold after the decrease in uridine nucleotides, when the accumulation of stable ribonucleic acid (RNA) stopped. The level of the nucleotide pool did not stabilize until 30 min after the change in optical density. The pool of dGTP dropped concomitantly with the pool of CTP. The nucleotide precursor PRPP exhibited a transient increase, wtih maximum value of four times the exponential levels at the onset of starvation. Apparently the cell adjusts early to starvation by reducing either the phosphorylating activity or the nucleotide biosynthetic activity. As in other downshift systems, the accumulation of stable RNA stopped before the break in optical density and before the stop in protein accumulation. Cell divisions were quite insensitive to the control mechanisms operating on RNA and protein accumulation under ammonium starvation, since the cells continued to divide for 21 min without any net accumulation of RNA. Images PMID:323222

Release abilities of adenosine diphosphate from phospholipid vesicles with different membrane properties and their hemostatic effects as a platelet substitute.

PubMed

Okamura, Yosuke; Katsuno, Shunsuke; Suzuki, Hidenori; Maruyama, Hitomi; Handa, Makoto; Ikeda, Yasuo; Takeoka, Shinji

2010-12-20

We have constructed phospholipid vesicles with hemostatic activity as a platelet substitute. The vesicles were conjugated with a dodecapeptide (HHLGGAKQAGDV, H12), which is a fibrinogen γ-chain carboxy-terminal sequence (γ400-411). We have recently exploited these vesicles as a potential drug delivery system by encapsulation of adenosine 5'-diphosphate (ADP) (H12-(ADP)-vesicles). Here we explore the relationship between the ADP release from H12-(ADP)-vesicles with different membrane properties and their hemostatic effects. In total, we prepared five kinds of H12-(ADP)-vesicles with different lamellarities and membrane flexibilities. By radioisotope-labeling, we directly show that H12-(ADP)-vesicles were capable of augmenting platelet aggregation by releasing ADP in an aggregation-dependent manner. The amount of ADP released from the vesicles was dependent on their membrane properties. Specifically, the amount of ADP released increased with decreasing lamellarity and tended to increase with increasing membrane flexibility. Our in vivo results clearly demonstrated that H12-(ADP)-vesicles with the ability to release ADP exert considerable hemostatic action in terms of correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rat model. We propose a recipe to control the hemostatic abilities of H12-(ADP)-vesicles by modulating ADP release based on membrane properties. We believe that this concept will be invaluable to the development of platelet substitutes and other drug carriers. Copyright © 2010 Elsevier B.V. All rights reserved.

Distribution of NTPDase5 and NTPDase6 and the regulation of P2Y receptor signalling in the rat cochlea

PubMed Central

O’Keeffe, Mary G.; Thorne, Peter R.; Housley, Gary D.; Robson, Simon C.

2010-01-01

Membrane-bound ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) in the inner ear regulate complex extracellular purinergic type-2 (P2) receptor signalling pathways through hydrolysis of extracellular nucleoside 5′-triphosphates and diphosphates. This study investigated the distribution of NTPDase5 and NTPDase6, two intracellular members of the E-NTPDase family, and linked this to regulation of P2 receptor signalling in the adult rat cochlea. These extracellular ectonucleotidases preferentially hydrolyse nucleoside 5′-diphosphates such as UDP and GDP. Expression of both enzymes at mRNA and protein level was detected in cochlear tissues and there was in vivo release of soluble NTPDase5 and 6 into cochlear fluids. Strong NTPDase5 immunostaining was found in the spiral ganglion neurones and supporting Deiters’ cells of the organ of Corti, while NTPDase6 was confined to the inner hair cells. Upregulation of NTPDase5 after exposure to loud sound indicates a dynamic role for NTPDase5 in cochlear response to stress, whereas NTPDase6 may have more limited extracellular roles. Noise-induced upregulation of co-localised UDP-preferring P2Y6 receptors in the spiral ganglion neurons further supports the involvement of NTPDase5 in regulation of P2Y receptor signalling. Noise stress also induced P2Y14 (UDP- and UDP-glucose preferring) receptor expression in the root processes of the outer sulcus cells, but this was not associated with localization of the E-NTPDases. PMID:20806016

Identification, functional characterization, and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in kiwifruit (Actinidia chinensis).

PubMed

Green, Sol A; Chen, Xiuyin; Nieuwenhuizen, Niels J; Matich, Adam J; Wang, Mindy Y; Bunn, Barry J; Yauk, Yar-Khing; Atkinson, Ross G

2012-03-01

Flowers of the kiwifruit species Actinidia chinensis produce a mixture of sesquiterpenes derived from farnesyl diphosphate (FDP) and monoterpenes derived from geranyl diphosphate (GDP). The tertiary sesquiterpene alcohol (E)-nerolidol was the major emitted volatile detected by headspace analysis. Contrastingly, in solvent extracts of the flowers, unusually high amounts of (E,E)-farnesol were observed, as well as lesser amounts of (E)-nerolidol, various farnesol and farnesal isomers, and linalool. Using a genomics-based approach, a single gene (AcNES1) was identified in an A. chinensis expressed sequence tag library that had significant homology to known floral terpene synthase enzymes. In vitro characterization of recombinant AcNES1 revealed it was an enzyme that could catalyse the conversion of FDP and GDP to the respective (E)-nerolidol and linalool terpene alcohols. Enantiomeric analysis of both AcNES1 products in vitro and floral terpenes in planta showed that (S)-(E)-nerolidol was the predominant enantiomer. Real-time PCR analysis indicated peak expression of AcNES1 correlated with peak (E)-nerolidol, but not linalool accumulation in flowers. This result, together with subcellular protein localization to the cytoplasm, indicated that AcNES1 was acting as a (S)-(E)-nerolidol synthase in A. chinensis flowers. The synthesis of high (E,E)-farnesol levels appears to compete for the available pool of FDP utilized by AcNES1 for sesquiterpene biosynthesis and hence strongly influences the accumulation and emission of (E)-nerolidol in A. chinensis flowers.

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene1[W

PubMed Central

Ghirardo, Andrea; Wright, Louwrance Peter; Bi, Zhen; Rosenkranz, Maaria; Pulido, Pablo; Rodríguez-Concepción, Manuel; Niinemets, Ülo; Brüggemann, Nicolas; Gershenzon, Jonathan; Schnitzler, Jörg-Peter

2014-01-01

The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties. PMID:24590857

The effect of cytidine-diphosphate choline (CDP-choline) on brain lipid changes during aging

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Medio, G.E.; Trovarelli, G.; Piccinin, G.L.

1984-01-01

Lipid synthesis has been tested in vivo in different brain areas of 12-month-old male rats. Cortex, striatum, brainstem, and subcortex of brain have been examined. The cerebellum was discarded. Mixtures of (2-/sup 3/H)glycerol and (Me-/sup 14/C)choline were injected into the lateral ventricle of the brain as lipid precursors, and their incorporation into total lipid, water-soluble intermediates and choline-containing phospholipids was examined 1 hr after isotope injection. In another series of experiments cytidine-5'-diphosphate choline (CDP-choline) was injected intraventricularly to the aged rats 10 min before sacrifice with a simultaneous injection, and radioactivity assays were performed as above. Distribution of radioactivity contentmore » of CDP-choline among brain areas 10 min after its administration showed a noticeable enrichment of the nucleotide and water-soluble-related compounds in the examined areas, but to a lesser degree in the cerebral cortex. The incorporation of labelled glycerol, which is severely depressed in aged rats in all four areas (Gaiti et al, 1982, 1983), was increased only in the cortex, and apparently decreased in the other areas. This last result is probably due to a dilution effect brought about by the administered cold CDP-choline upon the (/sup 14/C)-containing water-soluble metabolites. As a consequence, the (/sup 3/H)/(/sup 14/C) ratio in total lipid and in isolated phosphatidylcholine and choline plasmalogen increased after CDP-choline treatment.« less

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta.

PubMed

Gee, R.; Goyal, A.; Byerrum, R. U.; Tolbert, N. E.

1993-09-01

Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants.

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta.

PubMed Central

Gee, R.; Goyal, A.; Byerrum, R. U.; Tolbert, N. E.

1993-01-01

Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants. PMID:12231930

Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

PubMed

Wisitpitthaya, Somsinee; Zhao, Yi; Long, Marcus J C; Li, Minxing; Fletcher, Elaine A; Blessing, William A; Weiss, Robert S; Aye, Yimon

2016-07-15

The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2β2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families.

PubMed

Garcia, Mario D; Nouwens, Amanda; Lonhienne, Thierry G; Guddat, Luke W

2017-02-14

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted "S"-shaped conformation when bound to A. thaliana AHAS ( At AHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.