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Sample records for acetyl-coa synthases light

  1. Light- and Metabolism-related Regulation of the Chloroplast ATP Synthase Has Distinct Mechanisms and Functions*

    PubMed Central

    Kohzuma, Kaori; Dal Bosco, Cristina; Meurer, Jörg; Kramer, David M.

    2013-01-01

    The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification. PMID:23486473

  2. Mutation in cysteine bridge domain of the gamma-subunit affects light regulation of the ATP synthase in Arabidopsis

    USDA-ARS?s Scientific Manuscript database

    The chloroplast ATP synthase functions to synthesize ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine bridge in CF...

  3. The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

    PubMed

    Kohzuma, Kaori; Froehlich, John E; Davis, Geoffry A; Temple, Joshua A; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A; Kramer, David M

    2017-01-01

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

  4. Trehalose phosphate synthase overexpression in Parachlorella kessleri improves growth and photosynthetic performance under high light conditions.

    PubMed

    Rathod, Jayant Pralhad; Prakash, Gunjan; Vira, Chaitali; Lali, Arvind M

    2016-11-16

    Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.

  5. Light-Dependent Isoprene Emission (Characterization of a Thylakoid-Bound Isoprene Synthase in Salix discolor Chloroplasts).

    PubMed Central

    Wildermuth, M. C.; Fall, R.

    1996-01-01

    Isoprene synthase is an enzyme that is responsible for the production of the volatile C5 hydrocarbon, isoprene, in plant leaves. Isoprene formation in numerous C3 plants is interesting because (a) large quantities of isoprene are emitted, 5 x 1014 g of C annually, (b) a plant may release 1 to 8% of its fixed C as isoprene, and (c) the function of plant isoprene production is unknown. Because of the dependence of foliar isoprene emission on light, the existence of a plastidic isoprene synthase has been postulated. To pursue this idea, a method to isolate chloroplasts from Salix discolor was developed and shows a plastidic isoprene synthase that is tightly bound to the thylakoid membrane and accessible to trypsin inactivation. The thylakoid-bound isoprene synthase has catalytic properties similar to known soluble isoprene synthases; however, the relationship between these enzymes is unknown. The discovery of a thylakoid-bound isoprene synthase with a stromal-facing domain places it in the chloroplast, where it may be subject to numerous direct and indirect light-mediated effects. Implications for the light-dependent regulation of foliar isoprene production and its function are presented. PMID:12226383

  6. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  7. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  8. In situ localization of light-induced chalcone synthase mRNA, chalcone synthase, and flavonoid end products in epidermal cells of parsley leaves

    PubMed Central

    Schmelzer, Elmon; Jahnen, Willi; Hahlbrock, Klaus

    1988-01-01

    Methods involving in situ RNA hybridization, immunohistochemistry, and microspectrophotometry of individual cells were used to localize the mRNA encoding chalcone synthase (the key enzyme of flavonoid biosynthesis), the enzyme protein, and the biosynthetic end products in cross sections of parsley leaves (Petroselinum crispum). The light-dependent, sequential occurrence of all three components was restricted to epidermal cells. The results are in agreement with the putative function of flavonoids (transcriptionally inducible, UV-protective pigments) and suggest that all biosynthetic steps occur in those cells in which the products accumulate. Images PMID:16578833

  9. Differential roles of nitric oxide synthases in regulation of ultraviolet B light-induced apoptosis.

    PubMed

    Liu, Wei; Wu, Shiyong

    2010-11-01

    Ultraviolet B light (UVB) activates nitric oxide synthase(s) (NOSs) and nitric oxide (NO()) production, which plays a role in regulation of apoptosis. However, the role of NO() in UVB-induced apoptosis remains controversial. In this study, we analyzed expression and activation of constitutive NOSs (cNOSs) and their roles in UV-induced apoptosis of HaCaT keratinocytes. Our data showed that the expression of neuronal NOS (nNOS) was increased while endothelial NOS (eNOS) was uncoupled in the early phase (0-6 h) post-UVB. The expression of both cNOSs peaked at 12h post-UVB and NO() was transiently elevated with 30 min and then steadily rose from 6 to 18 h post-UVB. The expression of iNOS was detected at 6h post-UVB and then sturdily increased. Inhibition of cNOSs with L-NAME reduced the inducibility of NO(*) in the early and late phases of irradiation. Along with the eNOS uncoupling, an increased level of peroxynitrite (ONOO(-)) was detected in the early phase, but not in the late phase post-UVB. Inhibition of cNOSs reduced the production of ONOO(-) in the early time, but led to an increase of ONOO(-) in the late time after UVB-irradiation. The results indicate that cNOSs regulate NO()/ONOO(-) imbalance after UVB-irradiation. Our data suggested that the activation of cNOSs in the early phase post-UVB leads to NO()/ONOO(-) imbalance and promotes apoptosis via a caspase 3-independent pathway. The elevation of NO() in the late phase of UVB-irradiation is mainly produced by inducible NOS (iNOS). However, cNOSs also contribute to the NO() production and to maintain a higher NO()/ONOO(-) ratio, which reduces caspase 3 activity and protects cells from UVB-induced apoptosis.

  10. Light and Fungal Elicitor Induce 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase mRNA in Suspension Cultured Cells of Parsley (Petroselinum crispum L.) 1

    PubMed Central

    Henstrand, John M.; McCue, Kent F.; Brink, Kent; Handa, Avtar K.; Herrmann, Klaus M.; Conn, Eric E.

    1992-01-01

    Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated. ImagesFigure 1 PMID:16668708

  11. Light-dependent chlorophyll f synthase is a highly divergent paralog of PsbA of photosystem II.

    PubMed

    Ho, Ming-Yang; Shen, Gaozhong; Canniffe, Daniel P; Zhao, Chi; Bryant, Donald A

    2016-08-26

    Chlorophyll f (Chl f) permits some cyanobacteria to expand the spectral range for photosynthesis by absorbing far-red light. We used reverse genetics and heterologous expression to identify the enzyme for Chl f synthesis. Null mutants of "super-rogue" psbA4 genes, divergent paralogs of psbA genes encoding the D1 core subunit of photosystem II, abolished Chl f synthesis in two cyanobacteria that grow in far-red light. Heterologous expression of the psbA4 gene, which we rename chlF, enables Chl f biosynthesis in Synechococcus sp. PCC 7002. Because the reaction requires light, Chl f synthase is probably a photo-oxidoreductase that employs catalytically useful Chl a molecules, tyrosine YZ, and plastoquinone (as does photosystem II) but lacks a Mn4Ca1O5 cluster. Introduction of Chl f biosynthesis into crop plants could expand their ability to use solar energy.

  12. UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

    SciTech Connect

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Mishin, Vladimir; Laskin, Debra L.; Heck, Diane E.; Laskin, Jeffrey D.

    2008-10-01

    Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE{sub 2}, PGF{sub 2{alpha}}, PGD{sub 2} and PGI{sub 2} (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE{sub 2}, PGD{sub 2} and the PGD{sub 2} metabolite PGJ{sub 2}. Twenty-four hours after treatment with UVB (25 mJ/cm{sup 2}), production of PGE{sub 2} and PGJ{sub 2} increased, while PGD{sub 2} production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm{sup 2}) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE{sub 2} (EP1 and EP2), PGD{sub 2} (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.

  13. The sensitivity of light-evoked responses of retinal ganglion cells is decreased in nitric oxide synthase gene knockout mice.

    PubMed

    Wang, Guo-Yong; van der List, Deborah A; Nemargut, Joseph P; Coombs, Julie L; Chalupa, Leo M

    2007-11-30

    We have shown previously that increasing the production of nitric oxide (NO) results in a dampening of visual responses of retinal ganglion cells (G. Y. Wang, L. C. Liets, & L. M. Chalupa, 2003). To gain further insights into the role of NO in retinal function, we made whole-cell patch clamp recordings from ganglion cells of neural type nitric oxide synthase (nNOS) gene knockout mice. Here we show that in the dark-adapted state, the sensitivity of retinal ganglion cell to light stimulation is decreased in nNOS knockout animals. The lowest light intensities required to evoke optimal responses and the average intensities that evoked half-maximal responses were significantly higher in nNOS knockouts than in normal mice. Retinal histology and other features of light-evoked responses of ganglion cells in nNOS mice appeared to be indistinguishable from those of normal mice. Collectively, these results, in conjunction with our previous work, provide evidence that increasing levels of NO dampen visual responses of ganglion cells, while a lack of nNOS decreases the sensitivity of these neurons to light. Thus, NO levels in the retina are capable of modulating the information that ganglion cells convey to the visual centers of the brain.

  14. Endothelial nitric oxide synthase activation and nitric oxide function: new light through old windows.

    PubMed

    Bird, Ian M

    2011-09-01

    The principle mechanisms operating at the level of endothelial nitric oxide synthase (eNOS) itself to control its activity are phosphorylation, the auto-regulatory properties of the protein itself, and Ca(2)(+)/calmodulin binding. It is now clear that activation of eNOS is greatest when phosphorylation of certain serine and threonine residues is accompanied by elevation of cytosolic [Ca2+](i). While eNOS also contains an autoinhibitory loop, Rafikov et al. (2011) present the evidence for a newly identified 'flexible arm' that operates in response to redox state. Boeldt et al. (2011) also review the evidence that changes in the nature of endothelial Ca(2)(+) signaling itself in different physiologic states can extend both the amplitude and duration of NO output, and a failure to change these responses in pregnancy is associated with preeclampsia. The change in Ca(2)(+) signaling is mediated through altering capacitative entry mechanisms inherent in the cell, and so many agonist responses using this mechanism are altered. The term 'adaptive cell signaling' is also introduced for the first time to describe this phenomenon. Finally NO is classically regarded as a regulator of vascular function, but NO has other actions. One proposed role is regulation of steroid biosynthesis but the physiologic relevance was unclear. Ducsay & Myers (2011) now present new evidence that NO may provide the adrenal with a mechanism to regulate cortisol output according to exposure to hypoxia. One thing all three of these reviews show is that even after several decades of study into NO biosynthesis and function, there are clearly still many things left to discover.

  15. Size exclusion chromatography-multiangle laser light scattering analysis of hyaluronan size distributions made by membrane-bound hyaluronan synthase.

    PubMed

    Baggenstoss, Bruce A; Weigel, Paul H

    2006-05-15

    Size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analyses of Escherichia coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10-100 MDa) that coeluted with hyaluronan (HA) and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65-75 degrees C eliminated this artifact and increased the yield of recovered HA due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed uridine 5'-diphosphate (UDP) produced during the reaction, improved the linearity of HA synthesis-even at high substrate use. Surprisingly, the addition of EDTA, to chelate Mg(2+) ions, did not completely stop the HAS reaction at 30 degrees C or at 4 degrees C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4 to 6 MDa within 2-4 h.

  16. 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.

  17. Chalcone synthase family genes have redundant roles in anthocyanin biosynthesis and in response to blue/UV-A light in turnip (Brassica rapa; Brassicaceae).

    PubMed

    Zhou, Bo; Wang, Yu; Zhan, Yaguang; Li, Yuhua; Kawabata, Saneyuki

    2013-12-01

    The epidermis of Brassica rapa (turnip) cv. Tsuda contains light-induced anthocyanins, visible signs of activity of chalcone synthase (CHS), a key anthocyanin biosynthetic enzyme, which is encoded by the CHS gene family. To elucidate the regulation of this light-induced pigmentation, we isolated Brassica rapa CHS1-CHS6 (BrCHS1-CHS6) and characterized their cis-elements and expression patterns. Epidermises of light-exposed swollen hypocotyls (ESHS) were harvested to analyze transcription levels of BrCHS genes by real-time PCR. Different promoters for the genes were inserted into tobacco to examine pCHS-GUS activity by histochemistry. Yeast-one-hybridization was used to detect binding activity of BrCHS motifs to transcription factors. Transcript levels of BrCHS1, -4, and -5 and anthocyanin-biosynthesis-related genes F3H, DFR, and ANS were high, while those of BrCHS2, -3, and -6 were almost undetectable in pigmented ESHS. However, in leaves, CHS5, F3H, and ANS expression was higher than in nonpigmented ESHS, but transcription of DFR was not detected. In the analysis of BrCHS1 and BrCHS3 promoter activity, GUS activity was strong in pigmented flowers of BrPCHS1-GUS-transformed tobacco plants, but nearly absent in BrPCHS3-GUS-transformed plants. Transcript levels of regulators, BrMYB75 and BrTT8, were strongly associated with the anthocyanin content and were light-induced. Coregulated cis-elements were found in promoters of BrCHS1,-4, and -5, and BrMYB75 and BrTT8 had high binding activities to the BrCHS Unit 1 motif. The chalcone synthase gene family encodes a redundant set of light-responsive, tissue-specific genes that are expressed at different levels and are involved in flavonoid biosynthesis in Tsuda turnip.

  18. Arabidopsis S-Sulfocysteine Synthase Activity Is Essential for Chloroplast Function and Long-Day Light-Dependent Redox Control[W

    PubMed Central

    Bermúdez, Maria Angeles; Páez-Ochoa, Maria Angeles; Gotor, Cecilia; Romero, Luis C.

    2010-01-01

    In bacteria, the biosynthesis of Cys is accomplished by two enzymes that are encoded by the cysK and cysM genes. CysM is also able to use thiosulfate as a substrate to produce S-sulfocysteine. In plant cells, the biosynthesis of Cys occurs in the cytosol, mitochondria, and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes in Arabidopsis thaliana. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of CS26 suppressed the S-sulfocysteine synthase activity that was detected in the wild type; furthermore, the cs26 mutants exhibited a reduction in size and showed paleness, but penetrance of the growth phenotype depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants) as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify reactive oxygen species, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our findings reveal that S-sulfocysteine and the activity of S-sulfocysteine synthase play important roles in chloroplast function and are essential for light-dependent redox regulation within the chloroplast. PMID:20179139

  19. Cloning of a cDNA that encodes farnesyl diphosphate synthase and the blue-light-induced expression of the corresponding gene in the leaves of rice plants.

    PubMed

    Sanmiya, K; Iwasaki, T; Matsuoka, M; Miyao, M; Yamamoto, N

    1997-02-28

    A cDNA encoding farnesyl diphosphate synthase (FPPS), a key enzyme in isoprenoid biosynthesis, was isolated from a cDNA library constructed from mRNA that had been prepared from etiolated rice (Oriza sativa L. variety Nipponbare) seedlings after three hours of illumination by a subtraction method. The putative polypeptide deduced from the 1289 bp nucleotide sequence consisted of 353 amino acids and had a molecular mass of 40 676 Da. The predicted amino acid sequence exhibited high homology to those of FPPS from Arabidopsis (73% to type 1, 72% to type 2) and white lupin (74%). Southern blot analysis showed that the rice genome might contain only one gene for FPPS. The highest level of expression of the gene was demonstrated in leaves by RNA blot analysis. Moreover, light, in particular blue light, effectively enhanced expression of the gene.

  20. The role of constitutive nitric-oxide synthase in ultraviolet B light-induced nuclear factor κB activity.

    PubMed

    Tong, Lingying; Wu, Shiyong

    2014-09-19

    NF-κB is a transcription factor involved in many signaling pathways that also plays an important role in UV-induced skin tumorigenesis. UV radiation can activate NF-κB, but the detailed mechanism remains unclear. In this study, we provided evidence that the activation of constitutive nitric-oxide synthase plays a role in regulation of IκB reduction and NF-κB activation in human keratinocyte HaCaT cells in early phase (within 6 h) post-UVB. Treating the cells with l-NAME, a selective inhibitor of constitutive nitric-oxide synthase (cNOS), can partially reverse the IκB reduction and inhibit the DNA binding activity as well as nuclear translocation of NF-κB after UVB radiation. A luciferase reporter assay indicates that UVB-induced NF-κB activation is totally diminished in cNOS null cells. The cNOS-mediated reduction of IκB is likely due to the imbalance of nitric oxide/peroxynitrite because treating the cells with lower (50 μm), but not higher (100-500 μm), concentration of S-nitroso-N-acetylpenicillamine (SNAP) can reverse the effect of l-NAME in partial restore IκB level post-UVB. Our data also showed that NF-κB activity was required for maintaining a stable IκB kinase α subunit (IKKα) level because treating the cells with NF-κB or cNOS inhibitors could reduce IKKα level upon UVB radiation. In addition, our data demonstrated that although NF-κB protects cells from UVB-induced death, its pro-survival activity was likely neutralized by the pro-death activity of peroxynitrite after UVB radiation.

  1. Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent.

    PubMed

    Foresi, Noelia; Correa-Aragunde, Natalia; Parisi, Gustavo; Caló, Gonzalo; Salerno, Graciela; Lamattina, Lorenzo

    2010-11-01

    The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K(m) for the substrate l-Arg of 12 ± 5 μM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.

  2. Pretreatment with light-emitting diode therapy reduces ischemic brain injury in mice through endothelial nitric oxide synthase-dependent mechanisms.

    PubMed

    Lee, Hae In; Lee, Sae-Won; Kim, So Young; Kim, Nam Gyun; Park, Kyoung-Jun; Choi, Byung Tae; Shin, Yong-Il; Shin, Hwa Kyoung

    2017-03-24

    Photostimulation with low-level light emitting diode therapy (LED-T) modulates neurological and psychological functions. The purpose of this study was to evaluate the effects of LED-T pretreatment on the mouse brain after ischemia/reperfusion and to investigate the underlying mechanisms. Ischemia/reperfusion brain injury was induced by middle cerebral artery occlusion. The mice received LED-T twice a day for 2 days prior to cerebral ischemia. After reperfusion, the LED-T group showed significantly smaller infarct and edema volumes, fewer behavioral deficits compared to injured mice that did not receive LED-T and significantly higher cerebral blood flow compared to the vehicle group. We observed lower levels of endothelial nitric oxide synthase (eNOS) phosphorylation in the injured mouse brains, but significantly higher eNOS phosphorylation in LED-T-pretreated mice. The enhanced phospho-eNOS was inhibited by LY294002, indicating that the effects of LED-T on the ischemic brain could be attributed to the upregulation of eNOS phosphorylation through the phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, no reductions in infarct or edema volume were observed in LED-T-pretreated eNOS-deficient (eNOS(-/-)) mice. Collectively, we found that pretreatment with LED-T reduced the amount of ischemia-induced brain damage. Importantly, we revealed that these effects were mediated by the stimulation of eNOS phosphorylation via the PI3K/Akt pathway.

  3. Immunocytochemistry of endothelial nitric oxide synthase in the rat brain: a light and electron microscopical study using the tyramide signal amplification technique.

    PubMed

    Stanarius, A; Töpel, I; Schulz, S; Noack, H; Wolf, G

    1997-11-01

    There are many inconsistencies in the literature about the cellular and subcellular distribution of the endothelial isoform of nitric oxide synthase (eNOS) in the brain. We have re-investigated its localization by light and electron microscopical (LM, EM) immunocytochemistry and the NADPH-diaphorase reaction. Using bovine aortic tissue as a positive control the protocols for the fixation and staining procedure were optimized. Only cryosections immersion-fixed with aceton and a mixture of aldehydes exhibited a clear-cut immunostaining. In rat brain tissue the endothelium of the entire vasculature showed immunoreactivity and, in addition to that, the epithelial cells of the choroid plexuses, whereas neurons never displayed any signs of immunostaining. EM immunoprecipitates were seen irregularly distributed in the cytosol or attached to endocellular membranes. EM NADPH-diaphorase histochemistry using the tetrazolium salt BSPT provided incoherent pictures in so far as the reaction product was exclusively bound to membranes. The restriction of eNOS within brain tissue to the vasculature may have implications for the differential significance of NOS isoforms in brain function.

  4. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  5. The role of nitric-oxide synthase in the regulation of UVB light-induced phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

    PubMed

    Lu, Wei; László, Csaba F; Miao, Zhixin; Chen, Hao; Wu, Shiyong

    2009-09-04

    UV light induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2alpha in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2alpha phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2alpha phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2alpha after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2alpha phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N(G)-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2alpha phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2alpha phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.

  6. Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals

    PubMed Central

    Wu, Yisheng; Zhou, Ke; Toyomasu, Tomonobu; Sugawara, Chizu; Oku, Madoka; Abe, Shiho; Usui, Masami; Mitsuhashi, Wataru; Chono, Makiko; Chandler, Peter M.; Peters, Reuben J.

    2012-01-01

    Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals – specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1,2,&3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then revealed that TaCPS2 uniquely produces normal, rather than ent-, CPP; thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP synthase from rice (OsCPS4), while the non-inducible TaCPS3 and TaCPS4 cluster with the rice OsCPS1 required for gibberellin phytohormone biosynthesis, as well as with a barley (Hordeum vulgare) CPS (HvCPS1) that also is characterized here as similarly producing ent-CPP. These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here. PMID:23009878

  7. Lighting.

    SciTech Connect

    United States. Bonneville Power Administration.

    1992-09-01

    Since lighting accounts for about one-third of the energy used in commercial buildings, there is opportunity to conserve. There are two ways to reduce lighting energy use: modify lighting systems so that they used less electricity and/or reduce the number of hours the lights are used. This booklet presents a number of ways to do both. Topics covered include: reassessing lighting levels, reducing lighting levels, increasing bulb & fixture efficiency, using controls to regulate lighting, and taking advantage of daylight.

  8. Bacterial nitric oxide synthases.

    PubMed

    Crane, Brian R; Sudhamsu, Jawahar; Patel, Bhumit A

    2010-01-01

    Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. Despite some interesting differences in cofactor utilization and redox partners, the bacterial enzymes are in many ways similar to their mammalian NOS (mNOS) counterparts and, as such, have provided insight into the structural and catalytic properties of the NOS family. In particular, spectroscopic studies of thermostable bacterial NOSs have revealed key oxyheme intermediates involved in the oxidation of substrate L-arginine (Arg) to product NO. The biological functions of some bacterial NOSs have only more recently come to light. These studies disclose new roles for NO in biology, such as taking part in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage.

  9. Light

    NASA Astrophysics Data System (ADS)

    Vernon, C. G.

    2016-09-01

    Preface; 1. Historical; 2. Waves and wave-motion; 3. The behaviour of ripples; 4. The behaviour of light; 5. Refraction through glass blocks and prisms; 6. The imprinting of curvatures; 7. Simple mathematical treatment; 8. More advanced mathematical treatment; 9. The velocity of light; 10. The spectrum and colour; 11. Geometrical optics; 12. The eye and optical instruments; 13. Sources of light; 14. Interference, diffraction and polarisation; 15. Suggestions for class experiments; Index.

  10. Characterization of a Nitric Oxide Synthase from the Plant Kingdom: NO Generation from the Green Alga Ostreococcus tauri Is Light Irradiance and Growth Phase Dependent[C][W][OA

    PubMed Central

    Foresi, Noelia; Correa-Aragunde, Natalia; Parisi, Gustavo; Caló, Gonzalo; Salerno, Graciela; Lamattina, Lorenzo

    2010-01-01

    The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a Km for the substrate l-Arg of 12 ± 5 μM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology. PMID:21119059

  11. Maternal magnesium sulfate fetal neuroprotective effects to the fetus: inhibition of neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation in a rodent model.

    PubMed

    Beloosesky, Ron; Khatib, Nizar; Ginsberg, Yuval; Anabosy, Saja; Shalom-Paz, Einat; Dahis, Masha; Ross, Michael G; Weiner, Zeev

    2016-09-01

    Maternal magnesium administration has been shown to protect the preterm fetus from white- and gray-matter injury, although the mechanism is unknown. The purpose of the study is to test the following hypotheses: (1) maternal infections/inflammation activate fetal neuronal N-methyl-D-aspartate receptors that up-regulate neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells pathways; and (2) maternal magnesium sulfate attenuates fetal brain neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation through N-methyl-D-aspartate receptors. Pregnant rats at embryonic day 16 and embryonic day 18 (n = 6, 48 total) received injections of intraperitoneal lipopolysaccharide 500 μg/kg or saline at time 0. Dams were randomized for treatment with subcutaneous magnesium sulfate (270 mg/kg) or saline for 2 hours prior to and following lipopolysaccharide/saline injections. At 4 hours after lipopolysaccharide administration, fetal brains were collected from the 4 treatment groups (lipopolysaccharide/saline, lipopolysaccharide/magnesium sulfate, saline/magnesium sulfate, saline/saline), and phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and chemokine (C-C motif) ligand 2 protein levels were determined by Western blot. An additional group of pregnant rats (n = 5) received N-methyl-D-aspartate-receptor antagonist following the lipopolysaccharide injection to study magnesium sulfate protective mechanism. Lipopolysaccharide (lipopolysaccharide/saline) significantly increased fetal brain phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and chemokine (C-C motif) ligand 2 protein levels compared to the saline/saline group at both embryonic day 16 (phosphoneuronal nitric oxide synthase 0.23 ± 0.01 vs 0.11 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells

  12. Thioredoxin-insensitive plastid ATP synthase that performs moonlighting functions

    PubMed Central

    Kohzuma, Kaori; Dal Bosco, Cristina; Kanazawa, Atsuko; Dhingra, Amit; Nitschke, Wolfgang; Meurer, Jörg; Kramer, David M.

    2012-01-01

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory γ subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only γ1 is involved in ATP synthesis in photosynthesis. The γ1-containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only γ2-ATP synthase (gamma exchange-revised ATP synthase, gamera) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on γ2-ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in γ2 is substantially higher than that for γ1. This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that γ2-ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that γ2 originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses. PMID:22328157

  13. 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.

  14. 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.

  15. Nitric oxide synthase in tiger salamander retina.

    PubMed

    Kurenni, D E; Thurlow, G A; Turner, R W; Moroz, L L; Sharkey, K A; Barnes, S

    1995-10-23

    Previous studies have indicated that nitric oxide, a labile freely diffusible biological messenger synthesized by nitric oxide synthase, may modulate light transduction and signal transmission in the retina. In the present work, the large size of retinal cells in tiger salamander (Ambystoma tigrinum) allowed the utilization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and nitric oxide synthase immunocytochemistry to delineate the cell-specific intracellular localization of nitric oxide synthase. NADPH-diaphorase activity was highly concentrated in the outer retina, in rod and cone inner segment ellipsoids, and between and adjacent to the photoreceptor cell bodies in the outer nuclear layer. Examination of enzymatically isolated retinal cells indicated that outer nuclear layer NADPH-diaphorase activity was localized to the distal processes of the retinal glial (Müller) cells and to putative bipolar cell Landolt clubs. Less intense NADPH-diaphorase activity was seen in the photoreceptor inner segment myoid region, in a small number of inner nuclear layer cells, in cap-like configurations at the distal poles of cells in the ganglion cell layer and surrounding ganglion cell layer somata, and in punctate form within both plexiform layers, the pigment epithelium, and the optic nerve. Nitric oxide synthase-like immunoreactivity was similarly localized, but was also concentrated along a thin sublamina centered within the inner plexiform layer. The potential for nitric oxide generation at multiple retinal sites suggests that this molecule may play a number of roles in the processing of visual information in the retina.

  16. Lighting

    SciTech Connect

    Audin, L.

    1994-12-31

    EPAct covers a vast territory beyond lighting and, like all legislation, also contains numerous {open_quotes}favors,{close_quotes} compromises, and even some sleight-of-hand. Tucked away under Title XIX, for example, is an increase from 20% to 28% tax on gambling winnings, effective January 1, 1993 - apparently as a way to help pay for new spending listed elsewhere in the bill. Overall, it is a landmark piece of legislation, about a decade overdue. It remains to be seen how the Federal Government will enforce upgrading of state (or even their own) energy codes. There is no mention of funding for {open_quotes}energy police{close_quotes} in EPAct. Merely creating such a national standard, however, provides a target for those who sincerely wish to create an energy-efficient future.

  17. Purification and Characterization of Chorismate Synthase from Euglena gracilis 1

    PubMed Central

    Schaller, Andreas; van Afferden, Manfred; Windhofer, Volker; Bülow, Sven; Abel, Gernot; Schmid, Jürg; Amrhein, Nikolaus

    1991-01-01

    Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli. ImagesFigure 2Figure 3 PMID:16668543

  18. Hybrid polyketide synthases

    DOEpatents

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  19. Mammalian Ceramide Synthases

    PubMed Central

    Levy, Michal; Futerman, Anthony H.

    2010-01-01

    Summary In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their stucture and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases. PMID:20222015

  20. Mammalian ceramide synthases.

    PubMed

    Levy, Michal; Futerman, Anthony H

    2010-05-01

    In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their structure and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases.

  1. 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.

  2. The chloroplast ATP synthase features the characteristic redox regulation machinery.

    PubMed

    Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

    2013-11-20

    Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

  3. Biochemical Characterization of Stromal and Thylakoid-Bound Isoforms of Isoprene Synthase in Willow Leaves1

    PubMed Central

    Wildermuth, Mary C.; Fall, Ray

    1998-01-01

    Isoprene synthase is the enzyme responsible for the foliar emission of the hydrocarbon isoprene (2-methyl-1,3-butadiene) from many C3 plants. Previously, thylakoid-bound and soluble forms of isoprene synthase had been isolated separately, each from different plant species using different procedures. Here we describe the isolation of thylakoid-bound and soluble isoprene synthases from a single willow (Salix discolor L.) leaf-fractionation protocol. Willow leaf isoprene synthase appears to be plastidic, with whole-leaf and intact chloroplast fractionations yielding approximately equal soluble (i.e. stromal) and thylakoid-bound isoprene synthase activities. Although thylakoid-bound isoprene synthase is tightly bound to the thylakoid membrane (M.C. Wildermuth, R. Fall [1996] Plant Physiol 112: 171–182), it can be solubilized by pH 10.0 treatment. The solubilized thylakoid-bound and stromal isoprene synthases exhibit similar catalytic properties, and contain essential cysteine, histidine, and arginine residues, as do other isoprenoid synthases. In addition, two regulators of foliar isoprene emission, leaf age and light, do not alter the percentage of isoprene synthase activity in the bound or soluble form. The relationship between the isoprene synthase isoforms and the implications for function and regulation of isoprene production are discussed. PMID:9501144

  4. In Planta Recapitulation of Isoprene Synthases Evolution from Ocimene Synthases.

    PubMed

    Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

    2017-06-16

    Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated.We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated in vivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause in vivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms.Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Mechanisms of acetohydroxyacid synthases.

    PubMed

    Chipman, David M; Duggleby, Ronald G; Tittmann, Kai

    2005-10-01

    Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

  6. Isolation of streptococcal hyaluronate synthase.

    PubMed

    Prehm, P; Mausolf, A

    1986-05-01

    Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography.

  7. Isolation of streptococcal hyaluronate synthase.

    PubMed Central

    Prehm, P; Mausolf, A

    1986-01-01

    Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography. Images Fig. 1. Fig. 2. PMID:3092808

  8. Phosphanilic Acid Inhibits Dihydropteroate Synthase

    DTIC Science & Technology

    1989-11-01

    dihydropteroate synthases of P. aeruginosa and E . coli were about equally susceptible to inhibition by PA. These results suggest that cells of P. aeruginosa...are more permeable to PA than cells of E . coli . Although a weak inhibitor, PA acted on dihydropteroate synthase in the same manner as the sulfonamides...with which PA is structurally related. Inhibition of E . coli by PA in a basal salts-glucose medium was prevented by p-aminobenzoic acid (pABA). However

  9. RNA interference-mediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS.

    PubMed

    Cheng, Qiqing; Su, Ping; Hu, Yating; He, Yunfei; Gao, Wei; Huang, Luqi

    2014-02-01

    Tanshinones are a group of bioactive abietane-type norditerpenoid quinone compounds in Salvia miltiorrhiza. Copalyldiphosphate synthase of S. miltiorrhiza (SmCPS) is the first key enzyme in tanshinone biosynthesis from the universal diterpene precursor geranylgeranyl diphosphate. Hairy roots of S. miltiorrhiza were transformed with Agrobacterium rhizogenes carrying an RNA interference (RNAi) construct designed to silence SmCPS, and we examined the resulting SmCPS expression and tanshinone accumulation. In SmCPS–RNAi hairy roots, the transcript level of SmCPS was reduced to 26 % while the dihydrotanshinone I and cryptotanshinone levels were decreased by 53 and 38 % compared to those of the vector control hairy roots; tanshinone IIA was not detected. Therefore, the decreased expression of SmCPS caused a decrease in tanshinone levels which verifies that SmCPS is a key enzyme for tanshinone biosynthesis in S. miltiorrhiza.

  10. Lipoic Acid Synthase (LASY)

    PubMed Central

    Padmalayam, Indira; Hasham, Sumera; Saxena, Uday; Pillarisetti, Sivaram

    2009-01-01

    OBJECTIVE—Lipoic acid synthase (LASY) is the enzyme that is involved in the endogenous synthesis of lipoic acid, a potent mitochondrial antioxidant. The aim of this study was to study the role of LASY in type 2 diabetes. RESEARCH DESIGN AND METHODS—We studied expression of LASY in animal models of type 2 diabetes. We also looked at regulation of LASY in vitro under conditions that exist in diabetes. Additionally, we looked at effects of LASY knockdown on cellular antioxidant status, inflammation, mitochondrial function, and insulin-stimulated glucose uptake. RESULTS—LASY expression is significantly reduced in tissues from animal models of diabetes and obesity compared with age- and sex-matched controls. In vitro, LASY mRNA levels were decreased by the proinflammatory cytokine tumor necrosis factor (TNF)-α and high glucose. Downregulation of the LASY gene by RNA interference (RNAi) reduced endogenous levels of lipoic acid, and the activities of critical components of the antioxidant defense network, increasing oxidative stress. Treatment with exogenous lipoic acid compensated for some of these defects. RNAi-mediated downregulation of LASY induced a significant loss of mitochondrial membrane potential and decreased insulin-stimulated glucose uptake in skeletal muscle cells. In endothelial cells, downregulation of LASY aggravated the inflammatory response that manifested as an increase in both basal and TNF-α–induced expression of the proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Overexpression of the LASY gene ameliorated the inflammatory response. CONCLUSIONS—Deficiency of LASY results in an overall disturbance in the antioxidant defense network, leading to increased inflammation, insulin resistance, and mitochondrial dysfunction. PMID:19074983

  11. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    PubMed

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  12. Sucrose Synthase: Expanding Protein Function

    USDA-ARS?s Scientific Manuscript database

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  13. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    PubMed Central

    Ternes, Philipp; Brouwers, Jos F. H. M.; van den Dikkenberg, Joep; Holthuis, Joost C. M.

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually. PMID:19454763

  14. Submitochondrial localization, cell-free synthesis, and mitochondrial import of 2-isopropylmalate synthase of yeast.

    PubMed

    Hampsey, D M; Lewin, A S; Kohlhaw, G B

    1983-03-01

    2-Isopropylmalate synthase (EC 4.1.3.12) of yeast is a mitochondrial enzyme. We now provide evidence showing that a large part of the 2-isopropylmalate synthase activity that is associated with the mitochondria is located in the mitochondrial matrix. In vitro translation of total yeast RNA followed by immunoprecipitation with anti-2-isopropylmalate synthase antibody yields two polypeptides. The larger of these has an apparent molecular weight identical to that of purified 2-isopropylmalate synthase subunit (ca. 65,000). It is incorporated into isolated yeast mitochondria with no detectable change in molecular weight. The import requires energy. The smaller polypeptide migrates to a position corresponding to a molecular weight of 63,000-64,000. It is not taken up by mitochondria. Both polypeptides, which also can be obtained by immunoprecipitation of crude extracts, become labeled when in vitro translation is performed in the presence of N-formyl[35S]methionyl-tRNAf. Mutants with no detectable 2-isopropylmalate synthase activity are deficient in either one or both synthase-related polypeptides. These results are discussed in the light of recent evidence for two 2-isopropylmalate synthase-encoding genes in yeast.

  15. SIRT3 Deacetylates Ceramide Synthases

    PubMed Central

    Novgorodov, Sergei A.; Riley, Christopher L.; Keffler, Jarryd A.; Yu, Jin; Kindy, Mark S.; Macklin, Wendy B.; Lombard, David B.; Gudz, Tatyana I.

    2016-01-01

    Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2, and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2, and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, -2, and -6 are hyperacetylated in the mitochondria of SIRT3-null mice, and SIRT3 directly deacetylates the ceramide synthases in a NAD+-dependent manner that increases enzyme activity. Investigation of the SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron transport chain at the level of complex III, attenuated mitochondrial outer membrane permeabilization, and decreased reactive oxygen species generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke. PMID:26620563

  16. The leaf extract of Siberian Crabapple (Malus baccata (Linn.) Borkh) contains potential fatty acid synthase inhibitors.

    PubMed

    Wei, Xiang; Zhao, Ran; Sun, Ying-Hui; Cong, Jian-Ping; Meng, Fan-Guo; Zhou, Hai-Meng

    2009-02-01

    The present work focused on the kinetics of the inhibitory effects of the leaf extract of Siberian Crabapple, named Shan jingzi in China, on chicken liver fatty acid synthase. The results showed that this extract had much stronger inhibitory ability on fatty acid synthase than that from green teas described in many previous reports. The inhibitory ability of this extract is closely related to the extracting solvent, and the time of extraction was also an important influencing factor. The inhibitory types of this extract on diffeerent substrates of chicken liver fatty acid synthase, acetyl-CoA, malonyl-CoA and NADPH, were found to be noncompetitive, uncompetitive and mixed, respectively. The studies here shed a new light on the exploration for inhibitors of fatty acid synthase.

  17. Acetohydroxyacid synthases: evolution, structure, and function.

    PubMed

    Liu, Yadi; Li, Yanyan; Wang, Xiaoyuan

    2016-10-01

    Acetohydroxyacid synthase, a thiamine diphosphate-dependent enzyme, can condense either two pyruvate molecules to form acetolactate for synthesizing L-valine and L-leucine or pyruvate with 2-ketobutyrate to form acetohydroxybutyrate for synthesizing L-isoleucine. Because the key reaction catalyzed by acetohydroxyacid synthase in the biosynthetic pathways of branched-chain amino acids exists in plants, fungi, archaea, and bacteria, but not in animals, acetohydroxyacid synthase becomes a potential target for developing novel herbicides and antimicrobial compounds. In this article, the evolution, structure, and catalytic mechanism of acetohydroxyacid synthase are summarized.

  18. Shedding of hyaluronate synthase from streptococci.

    PubMed

    Mausolf, A; Jungmann, J; Robenek, H; Prehm, P

    1990-04-01

    Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state.

  19. Producing biofuels using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  20. Polyester synthases: natural catalysts for plastics.

    PubMed Central

    Rehm, Bernd H A

    2003-01-01

    Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with

  1. EXPRESSION OF THE GEOSMIN SYNTHASE GENE IN THE CYANOBACTERIUM ANABAENA CIRCINALIS AWQC318(1).

    PubMed

    Giglio, Steven; Saint, Christopher P; Monis, Paul T

    2011-12-01

    The occurrence of taste and odor episodes attributed to geosmin continues to trouble water utilities worldwide, and only recently have advances been made in our fundamental understanding of the biochemical and genetic mechanisms responsible for the production of geosmin in microorganisms. For the first time, we have examined the expression of the geosmin synthase gene and corresponding geosmin production by Anabaena circinalis Rabenh. ex Bornet et Flahault AWQC318 under conditions of continuous light illumination and the removal of light as a stimulus and demonstrate that the expression of geosmin synthase appears to be constitutive under these conditions. The decrease in geosmin synthase transcription post maximum cell numbers and stationary phase suggests that a decrease in isoprenoid synthesis may occur before a decrease in the transcription of ribosomal units as the process of cell death is initiated.

  2. Concerted versus Stepwise Mechanism in Thymidylate Synthase

    PubMed Central

    2015-01-01

    Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design. PMID:24949852

  3. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    PubMed

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  4. Cell wall polysaccharide synthases are located in detergent-resistant membrane microdomains in oomycetes.

    PubMed

    Briolay, Anne; Bouzenzana, Jamel; Guichardant, Michel; Deshayes, Christian; Sindt, Nicolas; Bessueille, Laurence; Bulone, Vincent

    2009-04-01

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1-->3)-beta-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1-->3)-beta-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

  5. Crystal structure of riboflavin synthase

    SciTech Connect

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  6. Postillumination Isoprene Emission: In Vivo Measurements of Dimethylallyldiphosphate Pool Size and Isoprene Synthase Kinetics in Aspen Leaves1

    PubMed Central

    Rasulov, Bahtijor; Copolovici, Lucian; Laisk, Agu; Niinemets, Ülo

    2009-01-01

    The control of foliar isoprene emission is shared between the activity of isoprene synthase, the terminal enzyme catalyzing isoprene formation from dimethylallyldiphosphate (DMADP), and the pool size of DMADP. Due to limited in vivo information of isoprene synthase kinetic characteristics and DMADP pool sizes, the relative importance of these controls is under debate. In this study, the phenomenon of postillumination isoprene release was employed to develop an in vivo method for estimation of the DMADP pool size and to determine isoprene synthase kinetic characteristics in hybrid aspen (Populus tremula × Populus tremuloides) leaves. The method is based on observations that after switching off the light, isoprene emission continues for 250 to 300 s and that the integral of the postillumination isoprene emission is strongly correlated with the isoprene emission rate before leaf darkening, thus quantitatively estimating the DMADP pool size associated with leaf isoprene emission. In vitro estimates demonstrated that overall leaf DMADP pool was very large, almost an order of magnitude larger than the in vivo pool. Yet, the difference between total DMADP pools in light and in darkness (light-dependent DMADP pool) was tightly correlated with the in vivo estimates of the DMADP pool size that is responsible for isoprene emission. Variation in in vivo DMADP pool size was obtained by varying light intensity and atmospheric CO2 and O2 concentrations. From these experiments, the in vivo kinetic constants of isoprene synthase were determined. In vivo isoprene synthase kinetic characteristics suggested that isoprene synthase mainly operates under substrate limitation and that short-term light, CO2, and O2 dependencies of isoprene emission result from variation in DMADP pool size rather than from modifications in isoprene synthase activity. PMID:19129417

  7. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana

    SciTech Connect

    Hemleben, V.; Frey, M.; Rall, S.; Koch, M.; Kittel, M.; Kreuzaler, F.; Ragg, H.; Fautz, E.; Hahlbrock, K.

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  8. The chloroplast ATP synthase: structural changes during catalysis.

    PubMed

    Richter, M L; Gao, F

    1996-10-01

    This article summarizes some of the evidence for the existence of light-driven structural changes in the epsilon and gamma subunits of the chloroplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a changed in the position of the epsilon subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modify epsilon Lys109; (2) enhanced solvent accessibility of several sulfhydryl residues on the gamma subunit; and (3) release/exchange of tightly bound ADP from the enzyme. Theses and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of the epsilon subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.

  9. Shedding of hyaluronate synthase from streptococci.

    PubMed Central

    Mausolf, A; Jungmann, J; Robenek, H; Prehm, P

    1990-01-01

    Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state. Images Fig. 2. Fig. 3. Fig. 5. PMID:2109602

  10. Genetics Home Reference: GM3 synthase deficiency

    MedlinePlus

    ... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... Testing (1 link) Genetic Testing Registry: Amish infantile epilepsy syndrome Other Diagnosis and Management Resources (2 links) ...

  11. Chitin synthase inhibitors as antifungal agents.

    PubMed

    Chaudhary, Preeti M; Tupe, Santosh G; Deshpande, Mukund V

    2013-02-01

    Increased risk of fungal diseases in immunocompromised patients, emerging fungal pathogens, limited repertoire of antifungal drugs and resistance development against the drugs demands for development of new and effective antifungal agents. With greater knowledge of fungal metabolism efforts are being made to inhibit specific enzymes involved in different biochemical pathways for the development of antifungal drugs. Chitin synthase is one such promising target as it is absent in plants and mammals. Nikkomycin Z, a chitin synthase inhibitor is under clinical development. Chitin synthesis in fungi, chitin synthase as a target for antifungal agent development, different chitin synthase inhibitors isolated from natural sources, randomly synthesized and modified from nikkomycin and polyoxin are discussed in this review.

  12. Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur-arene interaction as key to catalysis.

    PubMed

    Sun, Shi-Jing; Horikawa, Yoshiki; Wada, Masahisa; Sugiyama, Junji; Imai, Tomoya

    2016-11-03

    Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherichia coli cells that we recently constructed (cellulose-synthesizing E. coli, CESEC). We demonstrated that inactivating mutations at an important amino acid residue reduced cellulose production. In this study, an interesting loss-of-function mutation occurred on Cys308, whose main chain carbonyl plays an important role for locating the cellulose terminus. Mutating this cysteine to serine, thus changing sulfur to oxygen in the side chain, abolished cellulose production in addition to other apparent detrimental mutations. This unexpected result highlights that the thiol side-chain of this cysteine plays an active role in catalysis, and additional mutation experiments indicated that the sulfur-arene interaction around Cys308 is a key in cellulose-synthesizing activity. Data obtained by CESEC shed light on the function of cellulose synthase in living cells, and will deepen our understanding of the mechanism of cellulose synthase. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Terpene synthases from Cannabis sativa

    PubMed Central

    Booth, Judith K.; Page, Jonathan E.

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties. PMID:28355238

  14. Terpene synthases from Cannabis sativa.

    PubMed

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  15. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning Fo portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil γ-subunit. This acts as a rotating `cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the γ shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility - supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump - can also be explained by our model.

  16. Identification of avian wax synthases

    PubMed Central

    2012-01-01

    Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms. Results Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. Conclusions We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities. PMID:22305293

  17. Malate synthase a membrane protein

    SciTech Connect

    Chapman, K.D.; Turley, R.B.; Hermerath, C.A.; Carrapico, F.; Trelease, R.N.

    1987-04-01

    Malate synthase (MS) is generally regarded as a peripheral membrane protein, and believed by some to be ontogenetically associated with ER. However, immuno- and cyto-chemical in situ localizations show MS throughout the matrix of cotton (and cucumber) glyoxysomes, not specifically near their boundary membranes, nor in ER. Only a maximum of 50% MS can be solubilized from cotton glyoxysomes with 1% Triton X-100, 2mM Zwittergen 14, or 10mM DOC +/- salts. Cotton MS does not incorporate /sup 3/H-glucosamine in vivo, nor does it react with Con A on columns or blots. Cotton MS banded with ER in sucrose gradients (20-40%) in Tricine after 3h, but not after 22h in Tricine or Hepes, or after 3h in Hepes or K-phosphate. Collectively the authors data are inconsistent with physiologically meaningful MS-membrane associations in ER or glyoxysomes. It appears that experimentally-induced aggregates of MS migrate in ER gradients and occur in isolated glyoxysomes. These data indicate that ER is not involved in synthesis or modification of cottonseed MS prior to its import into the glyoxysomal matrix.

  18. Assay of Deoxyhypusine Synthase Activity

    PubMed Central

    Wolff, Edith C.; Lee, Seung Bum; Park, Myung Hee

    2011-01-01

    Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (<50 amino acids). Optimum conditions for the reaction and four detection methods for the product, deoxyhypusine-containing eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [3H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [3H] deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [3H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter. PMID:21318875

  19. Inhibitors of specific ceramide synthases.

    PubMed

    Schiffmann, Susanne; Hartmann, Daniela; Fuchs, Sina; Birod, Kerstin; Ferreiròs, Nerea; Schreiber, Yannick; Zivkovic, Aleksandra; Geisslinger, Gerd; Grösch, Sabine; Stark, Holger

    2012-02-01

    Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed.

  20. Isolation of fast purine nucleotide synthase ribozymes.

    PubMed

    Lau, Matthew W L; Cadieux, Kelly E C; Unrau, Peter J

    2004-12-08

    Here we report the in vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6-thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine ((6S)Gua). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M(-1) min(-1) and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a K(m) of approximately 80 muM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an 'RNA World' and provides a context from which to study small molecule RNA catalysis.

  1. Unique animal prenyltransferase with monoterpene synthase activity

    NASA Astrophysics Data System (ADS)

    Gilg, Anna B.; Tittiger, Claus; Blomquist, Gary J.

    2009-06-01

    Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.

  2. Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis.

    PubMed

    Johnson, J K; Srivastava, D K

    1991-06-01

    The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.

  3. The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    PubMed Central

    Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; Temple, Joshua A.; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A.; Kramer, David M.

    2017-01-01

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

  4. The Fused Anthranilate Synthase from Streptomyces venezuelae Functions as a Monomer

    PubMed Central

    Ashenafi, Meseret; Reddy, Prasad T.; Parsons, James F.; Byrnes, W. Malcolm

    2015-01-01

    Recently we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 μM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant kcat of 0.57 ± 0.02 s−1 at 30°C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but noncooperatively inhibited by tryptophan (Ki = 11.1 ± 0.1 μM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme’s minimal functional unit as a single TrpE-TrpG pair. PMID:25355158

  5. The fused anthranilate synthase from Streptomyces venezuelae functions as a monomer.

    PubMed

    Ashenafi, Meseret; Reddy, Prasad T; Parsons, James F; Byrnes, W Malcolm

    2015-02-01

    Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 μM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s(-1) at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 μM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme's minimal functional unit as a single TrpE-TrpG pair.

  6. Terpene synthases are widely distributed in bacteria

    PubMed Central

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  7. Polyethylene glycol-induced heteroassociation of malate dehydrogenase and citrate synthase

    SciTech Connect

    Merz, J.M.; Webster, T.A.; Appleman, J.R.; Manley, E.R.; Yu, H.A.; Datta, A.; Ackerson, B.J.; Spivey, H.O.

    1987-10-01

    Studies by dynamic and total intensity light scattering, ultracentrifugation, electron microscopy, and chemical crosslinking on solutions of the pig heart mitochondrial enzymes, malate dehydrogenase and citrate synthase (separately and together) demonstrate that polyethylene glycol induces very large homoassociations of each enzyme, and still larger heteroenzyme complexes between these two enzymes in the solution phase. Specificity of this heteroassociation is indicated by the facts that heteroassociations with bovine serum albumin were not observed for either the mitochondrial dehydrogenase or the synthase or between cytosolic malate dehydrogenase and citrate synthase. The weight fraction of the enzymes in the mitochondrial dehydrogenase-synthase associated particles in the solution phase was less than 0.03% with the dilute conditions used in the dynamic light scattering measurements. Neither palmitoyl-CoA nor other solution conditions tested significantly increased this weight fraction of associated enzymes in the solution phase. Because of the extremely low solubility of the associated species, however, the majority of the enzymes can be precipitated as the heteroenzyme complex. This precipitation is a classical first-order transition in spite of the large particle sizes and broad size distribution. Ionic effects on the solubility of the heteroenzyme complex appear to be of general electrostatic nature. Polyethylene glycol was found to be more potent in precipitating this complex than dextrans, polyvinylpyrrolidones, ficoll, and beta-lactoglobulin.

  8. Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

    PubMed Central

    Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

    2011-01-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

  9. Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules.

    PubMed

    Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C; Cresti, Mauro

    2011-03-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.

  10. A point mutation in atpC1 raises the redox potential of the Arabidopsis chloroplast ATP synthase gamma-subunit regulatory disulfide above the range of thioredoxin modulation

    USDA-ARS?s Scientific Manuscript database

    The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill-defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. T...

  11. Allene oxide synthases and allene oxides.

    PubMed

    Tijet, Nathalie; Brash, Alan R

    2002-08-01

    Allene oxides are unstable epoxides formed by the enzymatic dehydration of the lipoxygenase products of polyunsaturated fatty acids. The allene oxide synthases are of two structurally-unrelated types. In plants, a subfamily of cytochromes P450, designated as CYP74A, use the hydroperoxides of linoleic and linolenic acids as substrate. Both the 9- and 13-hydroperoxides may be converted to allene oxides and subsequently give rise to plant signaling molecules. In corals, a catalase-related hemoprotein functions as the allene oxide synthase. These marine invertebrates, as well as starfish, form allene oxides from the 8R-hydroperoxide of arachidonic acid. The coral allene oxide synthase from Plexaura homomalla occurs as the N-terminal domain of a natural fusion protein with the 8R-lipoxygenase that forms its substrate. This enzyme may be involved in biosynthesis of the cyclopentenone eicosanoids such as the clavulones.

  12. The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    DOE PAGES

    Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; ...

    2017-07-24

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

  13. Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB.

    PubMed

    Ma, Suzanne M; Tang, Yi

    2007-06-01

    The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.

  14. Protons, the thylakoid membrane, and the chloroplast ATP synthase.

    PubMed

    Junge, W

    1989-01-01

    According to the chemiosmotic theory, proton pumps and ATP synthases are coupled by lateral proton flow through aqueous phases. Three long-standing challenges to this concept, all of which have been loosely subsumed under 'localized coupling' in the literature, were examined in the light of experiments carried out with thylakoids: (1) Nearest neighbor interaction between pumps and ATP synthases. Considering the large distances between photosystem II and CFoCF1, in stacked thylakoids this is a priori absent. (2) Enhanced proton diffusion along the surface of the membrane. This could not be substantiated for the outer side of the thylakoid membrane. Even for the interface between pure lipid and water, two laboratories have reported the absence of enhanced diffusion. (3) Localized proton ducts in the membrane. Intramembrane domains that can transiently trap protons do exist in thylakoid membranes, but because of their limited storage capacity for protons, they probably do not matter for photophosphorylation under continuous light. Seemingly in favor of localized proton ducts is the failure of a supposedly permeant buffer to enhance the onset lag of photophosphorylation. However, it was found that failure of some buffers and the ability of others in this respect were correlated with their failure/ability to quench pH transients in the thylakoid lumen, as predicted by the chemiosmotic theory. It was shown that the chemiosmotic concept is a fair approximation, even for narrow aqueous phases, as in stacked thylakoids. These are approximately isopotential, and protons are taken in by the ATP synthase straight from the lumen. The molecular mechanism by which F0F1 ATPases couple proton flow to ATP synthesis is still unknown. The threefold structural symmetry of the headpiece that, probably, finds a corollary in the channel portion of these enzymes appeals to the common wisdom that structural symmetry causes functional symmetry. "Rotation catalysis" has been proposed. It is

  15. Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.

    PubMed

    Sato, Tsutomu; Yamaga, Hiroaki; Kashima, Shoji; Murata, Yusuke; Shinada, Tetsuro; Nakano, Chiaki; Hoshino, Tsutomu

    2013-05-10

    Basic enzyme: The tetraprenyl-β-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-β-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene.

  16. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  17. Producing dicarboxylic acids using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  18. Lessons from 455 Fusarium polyketide synthases

    USDA-ARS?s Scientific Manuscript database

    In fungi, polyketide synthases (PKSs) synthesize a structurally diverse array of secondary metabolites (SMs) with a range of biological activities. The most studied SMs are toxic to animals and/or plants, alter plant growth, have beneficial pharmaceutical activities, and/or are brightly colored pigm...

  19. Multisite phosphorylation of spinach leaf sucrose-phosphate synthase

    SciTech Connect

    Huber, J.L.; Huber, S.C. )

    1990-05-01

    Spinach leaf sucrose-phosphate synthase is phosphorylated both in vivo and in vitro on serine residues. Phosphorylation of SPS in vivo yields twelve major phosphopeptides after a tryptic digest and two dimensional mapping. The in vivo labeling of three of these SPS P-peptides is reduced in illuminated leaves where the extracted enzyme is activated relative to that of dark leaves. Two of these inhibitory sites are phosphorylated as well when SPS is inactivated in vitro using ({sup 32}P)ATP. In vivo phosphorylation of two other sites is enhanced during mannose feeding of the leaves (in light or dark) which produces the highest activation state of SPS. Overall, the results confirm that light-dark regulation of SPS activity occurs as a result of regulatory seryl-phosphorylation and involves a balance between phosphorylation of sites which inhibit or stimulate activity. Regulation of the SPS protein kinase that inhibits activity is relatively unaffected by phosphate but inhibited by G1c 6-P (IC{sub 50}{approx}5 mM), which may explain the control of SPS activation state by light-dark signals.

  20. Multi-level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance.

    PubMed

    Carrillo, L Ruby; Froehlich, John E; Cruz, Jeffrey A; Savage, Linda J; Kramer, David M

    2016-09-01

    The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ-subunit through the ferredoxin-thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild-type levels as irradiance was increased. This effect was caused by an altered redox state of the γ-subunit under low, but not high, light. The low light-specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non-photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin-thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  1. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    PubMed

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  2. Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria.

    PubMed Central

    Miyake, M; Kataoka, K; Shirai, M; Asada, Y

    1997-01-01

    Poly-beta-hydroxybutyrate (PHB) synthesis in a cyanobacterium, Synechococcus sp. strain MA19, is controlled at the enzyme level and is dependent on the C/N balance in the culture medium. The control involves at least two enzymes. The first enzyme is PHB synthase. Little PHB synthase activity was detected in crude extracts from cells grown under nitrogen-sufficient conditions (MA19(+N)). The activity was detected exclusively in membrane fractions from nitrogen-deprived cells (MA19(-N)) under light but not dark conditions. The shift in the enzyme activity was insensitive to chloramphenicol, which suggests posttranslational activation. Acetyl phosphate activated PHB synthase in membrane fractions from MA19(+N). In vitro, the activation level of PHB synthase changed, depending on the concentration of acetyl phosphate. The second enzyme was phosphotransacetylase (EC 2.3.1.8), which catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate. The activity was detected in crude extracts from MA19(-N) but not in those from MA19(+N). The results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C/N balance and intracellular acetyl-CoA concentration. Acetyl phosphate probably acts as a signal of C/N balance affecting PHB metabolism in MA19. PMID:9260940

  3. Bili lights

    MedlinePlus

    Phototherapy for jaundice; Bilirubin - bili lights; Neonatal care - bili lights; Newborn care - bili lights ... Phototherapy involves shining fluorescent light from the bili lights on bare skin. A specific wavelength of light can break down bilirubin into a form that ...

  4. 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.

  5. Polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and thymidylate synthase (TYMS) in multiple myeloma risk.

    PubMed

    Lima, Carmen S P; Ortega, Manoela M; Ozelo, Margareth C; Araujo, Renato C; De Souza, Cármino A; Lorand-Metze, Irene; Annichino-Bizzacchi, Joyce M; Costa, Fernando F

    2008-03-01

    We tested whether the polymorphisms of the methylenetetrahydrofolate reductase gene, MTHFR C677T and A1298C, the methionine synthase gene, MTR A2756G, the methionine synthase reductase gene, MTRR A66G, and the thymidylate synthase gene, TYMS 2R-->3R, involved in folate and methionine metabolism, altered the risk for multiple myeloma (MM). Genomic DNA from 123MM patients and 188 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. The frequency of the MTR 2756 AG plus GG genotype was higher in patients than in controls (39.8% versus 23.4%, P=0.001). Individual carriers of the variant allele G had a 2.31 (95% CI: 1.38-3.87)-fold increased risk for MM compared with others. In contrast, similar frequencies of the MTHFR, the MTRR and the TYMS genotypes were seen in patients and controls. These results suggest, for the first time, a role for the MTR A2756G polymorphism in MM risk in our country, but should be confirmed by large-scale epidemiological studies with patients and controls age matched.

  6. Benzophenone synthase from Garcinia mangostana L. pericarps.

    PubMed

    Nualkaew, Natsajee; Morita, Hiroyuki; Shimokawa, Yoshihiko; Kinjo, Keishi; Kushiro, Tetsuo; De-Eknamkul, Wanchai; Ebizuka, Yutaka; Abe, Ikuro

    2012-05-01

    The cDNA of a benzophenone synthase (BPS), a type III polyketide synthase (PKS), was cloned and the recombinant protein expressed from the fruit pericarps of Garcinia mangostana L., which contains mainly prenylated xanthones. The obtained GmBPS showed an amino acid sequence identity of 77-78% with other plant BPSs belonging to the same family (Clusiaceae). The recombinant enzyme produced 2,4,6-trihydroxybenzophenone as the predominant product with benzoyl CoA as substrate. It also accepted other substrates, such as other plant PKSs, and used 1-3 molecules of malonyl CoA to form various phloroglucinol-type and polyketide lactone-type compounds. Thus, providing GmBPS with various substrates in vivo might redirect the xanthone biosynthetic pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Caffeine synthase and related methyltransferases in plants.

    PubMed

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  8. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  9. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  10. Threonine Synthase of Lemna paucicostata Hegelm. 6746

    PubMed Central

    Giovanelli, John; Veluthambi, K.; Thompson, Gregory A.; Mudd, S. Harvey; Datko, Anne H.

    1984-01-01

    Threonine synthase (TS) was purified approximately 40-fold from Lemna paucicostata, and some of its properties determined by use of a sensitive and specific assay. During the course of its purification, TS was separated from cystathionine γ-synthase, establishing the separate identity of these enzymes. Compared to cystathionine γ-synthase, TS is relatively insensitive to irreversible inhibition by propargylglycine (both in vitro and in vivo) and to gabaculine, vinylglycine, or cysteine in vitro. TS is highly specific for O-phospho-l-homoserine (OPH) and water (hydroxyl ion). Nucleophilic attack by hydroxyl ion is restricted to carbon-3 of OPH and proceeds sterospecifically to form threonine rather than allo-threonine. The Km for OPH, determined at saturating S-adenosylmethionine (AdoMet), is 2.2 to 6.9 micromolar, two orders of magnitude less than values reported for TS from other plant tissues. AdoMet markedly stimulates the enzyme in a reversible and cooperative manner, consistent with its proposed role in regulation of methionine biosynthesis. Cysteine (1 millimolar) caused a slight (26%) reversible inhibition of the enzyme. Activities of TS isolated from Lemna were inversely related to the methionine nutrition of the plants. Down-regulation of TS by methionine may help to limit the overproduction of threonine that could result from allosteric stimulation of the enzyme by AdoMet. No evidence was obtained for feedback inhibition, repression, or covalent modification of TS by threonine and/or isoleucine. PMID:16663833

  11. Structure of a modular polyketide synthase

    PubMed Central

    Dutta, Somnath; Whicher, Jonathan R.; Hansen, Douglas A.; Hale, Wendi A.; Chemler, Joseph A.; Congdon, Grady R.; Narayan, Alison R.; Håkansson, Kristina; Sherman, David H.; Smith, Janet L.

    2014-01-01

    Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases, has an architecture in which successive modules catalyze two-carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We employed electron cryo-microscopy to visualize a full-length module and determine sub-nanometer resolution 3D reconstructions that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intra-module carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time the structural basis for both intra-module and inter-module substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems. PMID:24965652

  12. Progress towards clinically useful aldosterone synthase inhibitors.

    PubMed

    Cerny, Matthew A

    2013-01-01

    Owing to the high degree of similarity between aldosterone synthase (CYP11B2) and cortisol synthase (CYP11B1), the design of selective inhibitors of one or the other of these two enzymes was, at one time, thought to be impossible. Through development of novel enzyme screening assays and significant medicinal chemistry efforts, highly potent inhibitors of CYP11B2 have been identified with selectivities approaching 1000-fold between the two enzymes. Many of these molecules also possess selectivity against other steroidogenic cytochromes P450 (e.g. CYP17A1 and CYP19A1) as well as hepatic drug metabolizing P450s. Though not as well developed or explored, inhibitors of CYP11B1, with selectivities approaching 50-fold, have also been identified. The therapeutic benefits of affecting the renin-angiotensin-aldosterone system have been well established with the therapeutically useful angiotensin-converting enzymes inhibitors, angiotensin receptor blockers, and mineralocorticoid receptor antagonists. Data regarding the additional benefits of an aldosterone synthase inhibitor (ASi) are beginning to emerge from animal models and human clinical trials. Despite great promise and much progress, additional challenges still exist in the path towards development of a therapeutically useful ASi.

  13. Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT

    PubMed Central

    Vicidomini, R; Di Giovanni, A; Petrizzo, A; Iannucci, L F; Benvenuto, G; Nagel, A C; Preiss, A; Furia, M

    2015-01-01

    Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called ‘undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell–cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the

  14. Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

    PubMed

    Vicidomini, R; Di Giovanni, A; Petrizzo, A; Iannucci, L F; Benvenuto, G; Nagel, A C; Preiss, A; Furia, M

    2015-03-26

    Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still

  15. Acute treatment of constant darkness increases the efficiency of ATP synthase in rat liver mitochondria.

    PubMed

    Ramírez-Iñiguez, Ana Laura; Ortiz, Genaro Gabriel; El Hafidi, Mohammed; Rincón-Sánchez, Ana Rosa; Macías-Rodríguez, Ernesto; Pacheco-Moisés, Fermín Paul

    2009-01-01

    The circadian oscillations of many physiological processes provide an endogenous temporal program for the adaptive synchronization of mammals to the fluctuating external world. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system. The aim of the present work was investigate the effect of acute treatment of constant darkness on mitochondrial ATP synthase activities and membrane fluidity in liver from male rat. We found that ATP synthase activity was not changed by the treatment. However ATPase activity and membrane fluidity were significantly diminished and pH gradient driven by ATP hydrolysis was incremented, in comparison from samples from rats kept on normal light/dark cycles. Additionally, the treatment of constant darkness diminishes the passive proton permeability of the inner mitochondrial membrane. In conclusion constant darkness induces a more efficient coupling between proton transport and catalysis, and increment the efficiency of the enzyme because the ratio of ATP synthase/ATPase activity was higher. These results exhibited the physiological adaptation of liver mitochondria to acute treatment of constant darkness in order to satisfy the cellular energy demand.

  16. Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum.

    PubMed

    Liu, Hui; Zheng, Zhiming; Wang, Peng; Gong, Guohong; Wang, Li; Zhao, Genhai

    2013-04-01

    Chitin synthases catalyze the formation of β-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.

  17. Nitric oxide synthase is induced in sporulation of Physarum polycephalum

    PubMed Central

    Golderer, Georg; Werner, Ernst R.; Leitner, Stefan; Gröbner, Peter; Werner-Felmayer, Gabriele

    2001-01-01

    The myxomycete Physarum polycephalum expresses a calcium-independent nitric oxide (NO) synthase (NOS) resembling the inducible NOS isoenzyme in mammals. We have now cloned and sequenced this, the first nonanimal NOS to be identified, showing that it shares < 39% amino acid identity with known NOSs but contains conserved binding motifs for all NOS cofactors. It lacks the sequence insert responsible for calcium dependence in the calcium-dependent NOS isoenzymes. NOS expression was strongly up-regulated in Physarum macroplasmodia during the 5-day starvation period needed to induce sporulation competence. Induction of both NOS and sporulation competence were inhibited by glucose, a growth signal and known repressor of sporulation, and by l-N6–(1-iminoethyl)-lysine (NIL), an inhibitor of inducible NOS. Sporulation, which is triggered after the starvation period by light exposure, was also prevented by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive guanylate cyclase. In addition, also expression of lig1, a sporulation-specific gene, was strongly attenuated by NIL or ODQ. 8-Bromo-cGMP, added 2 h before the light exposure, restored the capacity of NIL-treated macroplasmodia to express lig1 and to sporulate. This indicates that the second messenger used for NO signaling in sporulation of Physarum is cGMP and links this signaling pathway to expression of lig1. PMID:11358872

  18. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  19. SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum.

    PubMed

    Chooi, Yit-Heng; Muria-Gonzalez, Mariano Jordi; Mead, Oliver L; Solomon, Peter S

    2015-08-15

    Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 (SnPKS19), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA, is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans. The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta. Subsequent characterization of the ΔSnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.

  20. 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

  1. Construction of gene expression system in hop (Humulus lupulus) lupulin gland using valerophenone synthase promoter.

    PubMed

    Okada, Yukio; Saeki, Kazuo; Inaba, Akira; Suda, Narushi; Kaneko, Takafumi; Ito, Kazutoshi

    2003-09-01

    The promoter region of the valerophenone synthase (VPS) gene was isolated from hop (Humulus lupulus). VPS, a member of the chalcone synthase (CHS) super-family, catalyzes the biosynthesis reaction of the hop resin that significantly accumulates in the cone's secretory gland called the "lupulin gland". The typical H-box and G-box sequences, which exist in many plants' CHS promoters and act as cis-elements for tissue specificity, UV-light induction, etc., were not found in the isolated VPS promoter, although the H-box-like sequence (CCTTACC, CCTAACC) and the core sequence (ACGT) of the G-box were observed. The transformation experiment using the VPS promoter-UIDA gene fusion revealed that the promoter acts not only in the lupulin gland but also in the glands of leaf and stem. On the other hand, the VPS promoter activity was not induced by UV-irradiation.

  2. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    PubMed

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  3. Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

    PubMed

    Gray, Dennis W; Breneman, Steven R; Topper, Lauren A; Sharkey, Thomas D

    2011-06-10

    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 K(m) 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.

  4. Lighting: Green Light.

    ERIC Educational Resources Information Center

    Maniccia, Dorine

    2003-01-01

    Explains that by using sustainable (green) building practices, schools and universities can make their lighting systems more efficient, noting that embracing green design principles can help schools attract students. Discusses lighting-control technologies (occupancy sensing technology, daylighting technology, and scheduling based technologies),…

  5. Lighting: Green Light.

    ERIC Educational Resources Information Center

    Maniccia, Dorine

    2003-01-01

    Explains that by using sustainable (green) building practices, schools and universities can make their lighting systems more efficient, noting that embracing green design principles can help schools attract students. Discusses lighting-control technologies (occupancy sensing technology, daylighting technology, and scheduling based technologies),…

  6. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  7. Cellulose synthase interacting protein: a new factor in cellulose synthesis.

    PubMed

    Gu, Ying; Somerville, Chris

    2010-12-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities.

  8. Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

    PubMed

    Kumar, Santosh; Kempinski, Chase; Zhuang, Xun; Norris, Ayla; Mafu, Sibongile; Zi, Jiachen; Bell, Stephen A; Nybo, Stephen Eric; Kinison, Scott E; Jiang, Zuodong; Goklany, Sheba; Linscott, Kristin B; Chen, Xinlu; Jia, Qidong; Brown, Shoshana D; Bowman, John L; Babbitt, Patricia C; Peters, Reuben J; Chen, Feng; Chappell, Joe

    2016-10-01

    Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg(2+) binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases. © 2016 American Society of Plant Biologists. All rights reserved.

  9. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  10. Divinyl ether synthase gene and protein, and uses thereof

    DOEpatents

    Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  11. The Thylakoid Membrane Protein CGL160 Supports CF1CF0 ATP Synthase Accumulation in Arabidopsis thaliana

    PubMed Central

    Fristedt, Rikard; Martins, Nádia Figueira; Strenkert, Daniela; Clarke, Cornelia A.; Suchoszek, Monika; Thiele, Wolfram; Schöttler, Mark Aurel; Merchant, Sabeeha S.

    2015-01-01

    The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF1CF0 complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF1 sub-complex. We suggest therefore that it may facilitate the assembly of CF1 into the holocomplex. PMID:25835989

  12. Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

    DOE PAGES

    Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori; ...

    2017-05-03

    In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+) in cfqmore » results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

  13. Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

    PubMed Central

    Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori; Hoh, Donghee; Strand, Deserah D.; Sato-Cruz, Mio; Savage, Linda; Cruz, Jeffrey A.; Fisher, Nicholas; Froehlich, John E.; Kramer, David M.

    2017-01-01

    In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage. PMID:28515738

  14. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    PubMed Central

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  15. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis.

    PubMed

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  16. Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

    PubMed

    Hayashi, H; Huang, P; Inoue, K; Hiraoka, N; Ikeshiro, Y; Yazaki, K; Tanaka, S; Kushiro, T; Shibuya, M; Ebizuka, Y

    2001-12-01

    An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

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

    PubMed

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

    2016-03-15

    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.

  18. 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

  19. Evolution and function of phytochelatin synthases.

    PubMed

    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  20. Activities and regulation of peptidoglycan synthases.

    PubMed

    Egan, Alexander J F; Biboy, Jacob; van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-10-05

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein-protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein-protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

  1. Acetylation of prostaglandin synthase by aspirin.

    PubMed Central

    Roth, G J; Stanford, N; Majerus, P W

    1975-01-01

    When microsomes of sheep or bovine seminal vesicles are incubated with [acetyl-3H]aspirin (acetyl salicylic acid), 200 Ci/mol, we observe acetylation of a single protein, as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The protein has a molecular weight of 85,000 and corresponds to a similar acetylated protein found in the particulate fraction of aspirin-treated human platelets. The aspirin-mediated acetylation reaction proceeds with the same time course and at the same concentration as does the inhibition of prostaglandin synthase (cyclo-oxygenase) (EC 1.14.99.1; 8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase) by the drug. At 100 muM aspirin, 50% inhibition of prostaglandin synthase and 50% of maximal acetylation are observed after 15 min at 37 degrees. Furthermore, the substrate for cyclo-oxygenase, arachidonic acid, inhibits protein acetylation by aspirin at concentrations (50% inhibition at 10-30 muM) which correlate with the Michaelis constant of arachidonic acid as a substrate for cyclooxygenase. Arachidonic acid analogues and indomethacin inhibit the acetylation reaction in proportion to their effectiveness as cyclo-oxygenase inhibitors. The results suggest that aspirin acts as an active-site acetylating agent for the enzyme cyclo-oxygenase. This action of aspirin may account for its anti-inflammatory and anti-platelet action. PMID:810797

  2. Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L. 1

    PubMed Central

    Berger, Michael G.; Sprengart, Michael L.; Kusnan, Misri; Fock, Heinrich P.

    1986-01-01

    Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO2 and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, 15NH4Cl was injected into the central pith of stem sections. The kinetics of 15N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of 15N derived from 15NH4Cl was almost completely inhibited. Injections of amido-15N glutamine demonstrated a potential for 15N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism. PMID:16664820

  3. Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L.

    PubMed

    Berger, M G; Sprengart, M L; Kusnan, M; Fock, H P

    1986-06-01

    Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO(2) and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, (15)NH(4)Cl was injected into the central pith of stem sections. The kinetics of (15)N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of (15)N derived from (15)NH(4)Cl was almost completely inhibited. Injections of amido-(15)N glutamine demonstrated a potential for (15)N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.

  4. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

    SciTech Connect

    Huber, J.L.A.; Huber, S.C. )

    1989-04-01

    Protein phosphorylation has been identified as a mechanism for the light-dark regulation of spinach sucrose-phosphate synthase (SPS) activity, previously shown to involve some type of covalent modification of the enzyme. The 120 kD subunit of SPS in extracts of light-treated leaves was labeled with {sup 32}P in the presence of ({gamma}-{sup 32}P) ATP. In this in vitro system, {sup 32}P incorporation into light-activated SPS was dependent upon ATP and magnesium concentrations as well as time, and was closely paralleled by inactivation of the enzyme. The soluble protein kinase involved in the interconversion of SPS between activated and deactivated forms may be specific for SPS as it co-purifies with SPS during partial purification of the enzyme. The kinase appears not to be calcium activated and no evidence has been obtained for metabolite control of SPS phosphorylation/inactivation.

  5. Rows of ATP Synthase Dimers in Native Mitochondrial Inner Membranes

    PubMed Central

    Buzhynskyy, Nikolay; Sens, Pierre; Prima, Valerie; Sturgis, James N.; Scheuring, Simon

    2007-01-01

    The ATP synthase is a nanometric rotary machine that uses a transmembrane electrochemical gradient to form ATP. The structures of most components of the ATP synthase are known, and their organization has been elucidated. However, the supramolecular assembly of ATP synthases in biological membranes remains unknown. Here we show with submolecular resolution the organization of ATP synthases in the yeast mitochondrial inner membranes. The atomic force microscopy images we have obtained show how these molecules form dimers with characteristic 15 nm distance between the axes of their rotors through stereospecific interactions of the membrane embedded portions of their stators. A different interaction surface is responsible for the formation of rows of dimers. Such an organization elucidates the role of the ATP synthase in mitochondrial morphology. Some dimers have a different morphology with 10 nm stalk-to-stalk distance, in line with ATP synthases that are accessible to IF1 inhibition. Rotation torque compensation within ATP synthase dimers stabilizes the ATP synthase structure, in particular the stator-rotor interaction. PMID:17557793

  6. Subcellular localization and regulation of coenzyme A synthase.

    PubMed

    Zhyvoloup, Alexander; Nemazanyy, Ivan; Panasyuk, Ganna; Valovka, Taras; Fenton, Tim; Rebholz, Heike; Wang, Mong-Lien; Foxon, Richard; Lyzogubov, Valeriy; Usenko, Vasylij; Kyyamova, Ramziya; Gorbenko, Olena; Matsuka, Genadiy; Filonenko, Valeriy; Gout, Ivan T

    2003-12-12

    CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

  7. Argininosuccinate synthase: at the center of arginine metabolism.

    PubMed

    Haines, Ricci J; Pendleton, Laura C; Eichler, Duane C

    2011-01-01

    The levels of L-arginine, a cationic, semi-essential amino acid, are often controlled within a cell at the level of local availability through biosynthesis. The importance of this temporal and spatial control of cellular L-arginine is highlighted by the tissue specific roles of argininosuccinate synthase (argininosuccinate synthetase) (EC 6.3.4.5), as the rate-limiting step in the conversion of L-citrulline to L-arginine. Since its discovery, the function of argininosuccinate synthase has been linked almost exclusively to hepatic urea production despite the fact that alternative pathways involving argininosuccinate synthase were defined, such as its role in providing arginine for creatine and for polyamine biosynthesis. However, it was the discovery of nitric oxide that meaningfully extended our understanding of the metabolic importance of non-hepatic argininosuccinate synthase. Indeed, our knowledge of the number of tissues that manage distinct pools of arginine under the control of argininosuccinate synthase has expanded significantly.

  8. Ubiquitination and filamentous structure of cytidine triphosphate synthase

    PubMed Central

    Pai, Li-Mei; Wang, Pei-Yu; Lin, Wei-Cheng; Chakraborty, Archan; Yeh, Chau-Ting; Lin, Yu-Hung

    2016-01-01

    ABSTRACT Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5′-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure. PMID:27116391

  9. Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases

    PubMed Central

    Aaron, Julie A.; Christianson, David. W.

    2011-01-01

    Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster not only serves to bind and orient the flexible isoprenoid substrate in the precatalytic Michaelis complex, but it also triggers the departure of the diphosphate leaving group to generate a carbocation that initiates catalysis. Additional conserved hydrogen bond donors assist the metal cluster in this function. Crystal structure analysis reveals that the constellation of three metal ions required for terpenoid synthase catalysis is generally identical among all class I terpenoid synthases of known structure. PMID:21562622

  10. A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.

    PubMed

    Shin, Daniel; Frane, Nicole D; Brecht, Ryan M; Keeler, Jesse; Nagarajan, Rajesh

    2015-12-01

    Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-l-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well-documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA- and acyl-ACP-utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.

  11. Functional Contribution of Chorismate Synthase, Anthranilate Synthase, and Chorismate Mutase to Penetration Resistance in Barley-Powdery Mildew Interactions

    USDA-ARS?s Scientific Manuscript database

    Plant processes resulting from primary or secondary metabolism have been hypothesized to contribute to defense against microbial attack. Barley chorismate synthase (HvCS), anthranilate synthase alpha subunit 2 (HvASa2) and chorismate mutase 1 (HvCM1) occupy pivotal branch-points downstream of the s...

  12. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage.

    PubMed

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Komur, Baran; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  13. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    PubMed Central

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders. PMID:27382570

  14. Circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, feeding and activity in rats.

    PubMed

    Kamerman, Peter; Mitchell, Duncan; Laburn, Helen

    2002-02-01

    We have investigated whether there is circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, physical activity and feeding. We used nocturnally active Sprague-Dawley rats, housed at approximately 24 degrees C with a 12:12 h light:dark cycle (lights on 07:00 hours) and provided with food and water ad libitum. Nitric oxide synthesis was inhibited by intraperitoneal injection of the unspecific nitric oxide synthase inhibitor N-nitro- L-arginine methyl ester ( L-NAME, 100, 50, 25, 10 mg/kg), or the relatively selective inducible nitric oxide synthase inhibitor aminoguanidine (100, 50 mg/kg), during the day ( approximately 09:00 hours) or night ( approximately 21:00 hours). Body temperature and physical activity were measured using radiotelemetry, while food intake was calculated by weighing each animal's food before as well as 12 and 24 h after each injection. We found that daytime injection of L-NAME and aminoguanidine had no effect on daytime body temperature. However, daytime injection of both drugs did decrease nocturnal food intake ( P<0.05) and activity ( P<0.05). When injected at night, L-NAME reduced night-time body temperature ( P<0.01), activity ( P<0.05) and food intake ( P<0.05) in a dose-dependent manner, but night-time injection of aminoguanidine inhibited only night-time activity ( P<0.05). The effects of nitric oxide synthase inhibition on body temperature, feeding and activity therefore are primarily a consequence of inhibiting constitutively expressed nitric oxide synthase, and are subject to circadian variation.

  15. Conversion of anthranilate synthase into isochorismate synthase: implications for the evolution of chorismate-utilizing enzymes.

    PubMed

    Plach, Maximilian G; Löffler, Patrick; Merkl, Rainer; Sterner, Reinhard

    2015-09-14

    Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free-living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.

  16. Identification of cystathionine γ-synthase and threonine synthase from Cicer arietinum and Lens culinaris.

    PubMed

    Morneau, Dominique J K; Jaworski, Allison F; Aitken, Susan M

    2013-04-01

    In plants, cystathionine γ-synthase (CGS) and threonine synthase (TS) compete for the branch-point metabolite O-phospho-L-homoserine. These enzymes are potential targets for metabolic engineering studies, aiming to alter the flux through the competing methionine and threonine biosynthetic pathways, with the goal of increasing methionine production. Although CGS and TS have been characterized in the model organisms Escherichia coli and Arabidopsis thaliana, little information is available on these enzymes in other, particularly plant, species. The functional CGS and TS coding sequences from the grain legumes Cicer arietinum (chickpea) and Lens culinaris (lentil) identified in this study share approximately 80% amino acid sequence identity with the corresponding sequences from Glycine max. At least 7 active-site residues of grain legume CGS and TS are conserved in the model bacterial enzymes, including the catalytic base. Putative processing sites that remove the targeting sequence and result in functional TS were identified in the target species.

  17. Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase.

    PubMed

    Martin, Christine J; Timoney, Máire C; Sheridan, Rose M; Kendrew, Steven G; Wilkinson, Barrie; Staunton, James C; Leadlay, Peter F

    2003-12-07

    A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.

  18. Endothelial nitric oxide synthase in the microcirculation

    PubMed Central

    Shu, Xiaohong; Keller, T.C. Stevenson; Begandt, Daniela; Butcher, Joshua T.; Biwer, Lauren; Keller, Alexander S.; Columbus, Linda; Isakson, Brant E.

    2015-01-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO) - a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells. PMID:26390975

  19. Endothelial nitric oxide synthase in the microcirculation.

    PubMed

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  20. Kinetic studies on muscle glycogen synthase.

    PubMed

    Salsas, E; Larner, J

    1975-05-10

    Using the I form of rabbit muscle glycogen synthase essentially free of glycogen, the kinetics and mechanism of action was investigated. No evidence for an exchange between [14C]UDP and UDP-glucose was found. The bisubstrate kinetics of the enzyme for UDP-glucose and glycogen, as well as for UDP-glucose and maltose, was determined. An intersecting pattern in the double reciprocal plot (velocity versus substrate concentration) suggestive of a sequential mechanism (ordered or random) was found in all cases. The K-m for UDP-glucose (45 to 48 mM) was the same with either maltose or glycogen as acceptor. The K-m for maltose (230 mM) and for glycogen (1.5 mug/ml) differed.

  1. Inducible nitric oxide synthase: Good or bad?

    PubMed

    Lind, Maggie; Hayes, Alan; Caprnda, Martin; Petrovic, Daniel; Rodrigo, Luis; Kruzliak, Peter; Zulli, Anthony

    2017-09-01

    Nitric oxide synthases (NOS) are a family of isoforms responsible for the synthesis of the potent dilator nitric oxide (NO). Expression of inducible NOS (iNOS) occurs in conditions of inflammation, and produces large amounts of NO. In pathological conditions iNOS is regarded as a harmful enzyme and is proposed to be a major contributor to diseases of the cardiovascular system such as atherosclerosis. In this review, we address the notion that iNOS is a detrimental enzyme in disease and discuss its potentially beneficial roles. Additionally, we describe other molecules associated with iNOS in diseases such as atherosclerosis, and current research on therapeutic inhibitors tested to reduced pathology associated with cardiovascular diseases (CVD). Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Nitric Oxide Synthases and Atrial Fibrillation

    PubMed Central

    Bonilla, Ingrid M.; Sridhar, Arun; Györke, Sandor; Cardounel, Arturo J.; Carnes, Cynthia A.

    2012-01-01

    Oxidative stress has been implicated in the pathogenesis of atrial fibrillation. There are multiple systems in the myocardium which contribute to redox homeostasis, and loss of homeostasis can result in oxidative stress. Potential sources of oxidants include nitric oxide synthases (NOS), which normally produce nitric oxide in the heart. Two NOS isoforms (1 and 3) are normally expressed in the heart. During pathologies such as heart failure, there is induction of NOS 2 in multiple cell types in the myocardium. In certain conditions, the NOS enzymes may become uncoupled, shifting from production of nitric oxide to superoxide anion, a potent free radical and oxidant. Multiple lines of evidence suggest a role for NOS in the pathogenesis of atrial fibrillation. Therapeutic approaches to reduce atrial fibrillation by modulation of NOS activity may be beneficial, although further investigation of this strategy is needed. PMID:22536189

  3. The Rotary Mechanism of the ATP Synthase

    PubMed Central

    Nakamoto, Robert K.; Scanlon, Joanne A. Baylis; Al-Shawi, Marwan K.

    2008-01-01

    The FOF1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous FO sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. PMID:18515057

  4. Nuclear glycogen and glycogen synthase kinase 3.

    PubMed

    Ragano-Caracciolo, M; Berlin, W K; Miller, M W; Hanover, J A

    1998-08-19

    Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus. Copyright 1998 Academic Press.

  5. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    PubMed Central

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  6. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    PubMed Central

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds. PMID:19710020

  7. Enhanced gastric nitric oxide synthase activity in duodenal ulcer patients.

    PubMed Central

    Rachmilewitz, D; Karmeli, F; Eliakim, R; Stalnikowicz, R; Ackerman, Z; Amir, G; Stamler, J S

    1994-01-01

    Nitric oxide, the product of nitric oxide synthase in inflammatory cells, may have a role in tissue injury through its oxidative metabolism. Nitric oxide may have a role in the pathogenesis of duodenal ulcer and may be one of the mechanisms responsible for the association between gastric infection with Helicobacter pylori and peptic disease. In this study, calcium independent nitric oxide synthase activity was detected in human gastric mucosa suggesting expression of the inducible isoform. In 17 duodenal ulcer patients gastric antral and fundic nitric oxide synthase activity was found to be two and 1.5-fold respectively higher than its activity in the antrum and fundus of 14 normal subjects (p < 0.05). H pylori was detected in the antrum of 15 of 17 duodenal ulcer patients and only in 7 of 14 of the control subjects. Antral nitric oxide synthase activity in H pylori positive duodenal ulcer patients was twofold higher than in H pylori positive normal subjects (p < 0.05). In duodenal ulcer patients antral and fundic nitric oxide synthase activity resumed normal values after induction of ulcer healing with ranitidine. Eradication of H pylori did not further affect gastric nitric oxide synthase activity. These findings suggest that in duodenal ulcer patients stimulated gastric mucosal nitric oxide synthase activity, though independent of the H pylori state, may contribute to the pathogenesis of the disease. PMID:7525417

  8. Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors.

    PubMed Central

    Poole, M A; Homann, M J; Bae-Lee, M S; Carman, G M

    1986-01-01

    The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHO1) and the opi1 mutant. VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis. Images PMID:3023284

  9. CTP synthase forms cytoophidia in the cytoplasm and nucleus.

    PubMed

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. Copyright © 2014. Published by Elsevier Inc.

  10. Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus.

    PubMed

    Tsuge, Takeharu; Hyakutake, Manami; Mizuno, Kouhei

    2015-08-01

    This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

  11. ATP Synthase Deficiency due to TMEM70 Mutation Leads to Ultrastructural Mitochondrial Degeneration and Is Amenable to Treatment

    PubMed Central

    Braczynski, Anne K.; Vlaho, Stefan; Müller, Klaus; Wittig, Ilka; Blank, Anna-Eva; Tews, Dominique S.; Drott, Ulrich; Kleinle, Stephanie; Abicht, Angela; Horvath, Rita; Plate, Karl H.; Stenzel, Werner; Goebel, Hans H.; Schulze, Andreas; Harter, Patrick N.; Kieslich, Matthias; Mittelbronn, Michel

    2015-01-01

    TMEM70 is involved in the biogenesis of mitochondrial ATP synthase and mutations in the TMEM70 gene impair oxidative phosphorylation. Herein, we report on pathology and treatment of ATP synthase deficiency in four siblings. A consanguineous family of Roma (Gipsy) ethnic origin gave birth to 6 children of which 4 were affected presenting with dysmorphic features, failure to thrive, cardiomyopathy, metabolic crises, and 3-methylglutaconic aciduria as clinical symptoms. Genetic testing revealed a homozygous mutation (c.317-2A>G) in the TMEM70 gene. While light microscopy was unremarkable, ultrastructural investigation of muscle tissue revealed accumulation of swollen degenerated mitochondria with lipid crystalloid inclusions, cristae aggregation, and exocytosis of mitochondrial material. Biochemical analysis of mitochondrial complexes showed an almost complete ATP synthase deficiency. Despite harbouring the same mutation, the clinical outcome in the four siblings was different. Two children died within 60 h after birth; the other two had recurrent life-threatening metabolic crises but were successfully managed with supplementation of anaplerotic amino acids, lipids, and symptomatic treatment during metabolic crisis. In summary, TMEM70 mutations can cause distinct ultrastructural mitochondrial degeneration and almost complete deficiency of ATP synthase but are still amenable to treatment. PMID:26550569

  12. Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

    PubMed

    Bañuelos, O; Casqueiro, J; Steidl, S; Gutiérrez, S; Brakhage, A; Martín, J F

    2002-01-01

    There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

  13. Light Pollution

    ERIC Educational Resources Information Center

    Riegel, Kurt W.

    1973-01-01

    Outdoor lighting is light pollution which handicaps certain astronomical programs. Protective measures must be adopted by the government to aid observational astronomy without sacrificing legitimate outdoor lighting needs. (PS)

  14. Light Duty.

    ERIC Educational Resources Information Center

    Rogers, Jeff

    1996-01-01

    Discusses multipurpose athletic-field lighting specifications to enhance lighting quality and reduce costs. Topics discussed include lamp choice, lighting spillover and glare prevention, luminary assemblies and poles, and the electrical dimming and switching systems. (GR)

  15. Light Pollution

    ERIC Educational Resources Information Center

    Riegel, Kurt W.

    1973-01-01

    Outdoor lighting is light pollution which handicaps certain astronomical programs. Protective measures must be adopted by the government to aid observational astronomy without sacrificing legitimate outdoor lighting needs. (PS)

  16. Light Duty.

    ERIC Educational Resources Information Center

    Rogers, Jeff

    1996-01-01

    Discusses multipurpose athletic-field lighting specifications to enhance lighting quality and reduce costs. Topics discussed include lamp choice, lighting spillover and glare prevention, luminary assemblies and poles, and the electrical dimming and switching systems. (GR)

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

    SciTech Connect

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-02-26

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  18. Generation and Functional Evaluation of Designer Monoterpene Synthases.

    PubMed

    Srividya, N; Lange, I; Lange, B M

    2016-01-01

    Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes. © 2016 Elsevier Inc. All rights reserved.

  19. Peroxisomal and mitochondrial citrate synthase in CAM plants.

    PubMed

    Zafra, M F; Segovia, J L; Alejandre, M J; García-Peregrín, E

    1981-12-01

    Citrate synthase wa studied for the first time in peroxisomes and mitochondria of crassulacean acid metabolism plants. Cellular organelles were isolated from Agave americana leaves by sucrose density gradient centrifugation and characterized by the use of catalase and cytochrome oxidase as marker enzymes, respectively. 48,000 X g centrifugation caused the breakdown of the cellular organelles. The presence of a glyoxylate cycle enzyme (citrate synthase) and a glycollate pathway enzyme (catalase) in the same organelles, besides the absence of another glyoxalate cycle enzyme (malate synthase) is reported for the first time, suggesting that peroxisomal and glyoxysomal proteins are synthesized at the same time and housed in he same organelle.

  20. Glycogen synthase kinase 3 regulates photic signaling in the suprachiasmatic nucleus.

    PubMed

    Paul, Jodi R; McKeown, Alex S; Davis, Jennifer A; Totsch, Stacie K; Mintz, Eric M; Kraft, Timothy W; Cowell, Rita M; Gamble, Karen L

    2017-04-01

    Glycogen synthase kinase 3 (GSK3) is a serine-threonine kinase that regulates mammalian circadian rhythms at the behavioral, molecular and neurophysiological levels. In the central circadian pacemaker, the suprachiasmatic nucleus (SCN), inhibitory phosphorylation of GSK3 exhibits a rhythm across the 24 h day. We have recently shown that GSK3 is capable of influencing both the molecular clock and SCN neuronal activity rhythms. However, it is not known whether GSK3 regulates the response to environmental cues such as light. The goal of this study was to test the hypothesis that GSK3 activation mediates light-induced SCN excitability and photic entrainment. Immunofluorescence staining in the SCN of mice showed that late-night light exposure significantly increased GSK3 activity (decreased pGSK3β levels) 30-60 min after the light-pulse. In addition, pharmacological inhibition of GSK3 blocked the expected light-induced excitability in SCN neurons; however, this effect was not associated with changes in resting membrane potential or input resistance. Behaviorally, mice with constitutively active GSK3 (GSK3-KI) re-entrained to a 6-h phase advance in the light-dark cycle in significantly fewer days than WT control animals. Furthermore, the behavioral and SCN neuronal activity of GSK3-KI mice was phase-advanced compared to WT, in both normal and light-exposed conditions. Finally, GSK3-KI mice exhibited normal negative-masking behavior and electroretinographic responses to light, suggesting that the enhanced photic entrainment is not due to an overall increased sensitivity to light in these animals. Taken together, these results provide strong evidence that GSK3 activation contributes to light-induced phase-resetting at both the neurophysiological and behavioral levels. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  1. O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase

    PubMed Central

    Talukdar, Arindam; Zhao, Yujie; Lv, Wei; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Cushman, Mark

    2012-01-01

    Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction. PMID:22780198

  2. Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

    PubMed Central

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses. PMID:22645501

  3. Zinc affects differently growth, photosynthesis, antioxidant enzyme activities and phytochelatin synthase expression of four marine diatoms.

    PubMed

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

  4. Characterization of chitin synthases from Entamoeba.

    PubMed

    Campos-Góngora, Eduardo; Ebert, Frank; Willhoeft, Ute; Said-Fernández, S; Tannich, Egbert

    2004-09-01

    A major component of the Entamoeba cyst wall is chitin, a homopolymer of beta-(1,4)-linked N-acetyl-D-glucosamine. Polymerization of chitin requires the presence of active chitin synthases (CHS), a group of enzymes belonging to the family of beta-glycosyl transferases. CHS have been described for fungi, insects, and nematodes; however, information is lacking about the structure and expression of this class of enzymes in protozoons such as Entamoeba. In this study, the primary structures of two putative E. histolytica CHS (EhCHS-1 and EhCHS-2) were determined by gene cloning and homologous proteins were identified in databases from E. dispar and the reptilian parasite E. invadens. The latter constitutes the widely used model organism for the study of Entamoeba cyst development. The two ameba enzymes revealed between 23% and 33% sequence similarity to CHS from other organisms with full conservation of all residues critically important for CHS activity. Interestingly, EhCHS-1 and EhCHS-2 differed substantially in their predicted molecular weights (73 kD vs. 114 kD) as well as in their isoelectric points (5.04 vs. 8.05), and homology was restricted to a central stretch of about 400 amino acid residues containing the catalytic domain. Outside the catalytic domain, EhCHS-1 was predicted to have seven transmembrane helices (TMH) of which the majority is located within the C-terminal part, resembling the situation found in yeast; whereas, EhCHS-2 is structurally related to nematode or insect chitin synthases, as it contained 17 predicted TMHs of which the majority is located within the N-terminal part of the molecule. Northern blot analysis revealed that genes corresponding to CHS-1 and CHS-2 are not expressed in Entamoeba trophozoites, but substantial amounts of CHS-1 and CHS-2 RNA were present 4 to 8 hours after induction of cyst formation by glucose deprivation of E. invadens. The time-courses of expression differed slightly between the two ameba CHS genes, as in

  5. Rare structural variants of human and murine uroporphyrinogen I synthase

    SciTech Connect

    Meisler, M.H.; Carter, M.L.C.

    1980-05-01

    An isoelectric focusing method for detection of structural variants of the enzyme uroporphyrinogen I synthase (porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8) in mammalian tissues has been developed. Mouse and human erythrocytes contain one or two major isozymes of uroporphyrinogen I synthase, respectively. Other tissues contain a set of more acidic isozymes that are encoded by the same structural gene as the erythrocyte isozymes. Mouse populations studied with this method were monomorphic for uroporphyrinogen I synthase, with the exception of one feral mouse population. The pedigree of a human family with a rare structural variant is consistent with autosomal linkage of the structural gene. This system provides a convenient isozyme marker for genetic studies and will facilitate determination of the chromosomal location of the uroporphyrinogen I synthase locus.

  6. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    PubMed

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  7. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    PubMed Central

    Ahmad, Zulfiqar; Laughlin, Thomas F.; Kady, Ismail O.

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  8. Lighting Conservation

    ERIC Educational Resources Information Center

    Arnold, Frank D.

    1975-01-01

    With the energy crisis has come an awareness of wasteful consumption practices. One area where research is being done is in lighting conservation. Information in this article is concerned with finding more effective and efficient lighting designs which include daylight utilization, task-oriented lighting, and lighting controls. (MA)

  9. Myocardial Regulation of Lipidomic Flux by Cardiolipin Synthase

    PubMed Central

    Kiebish, Michael A.; Yang, Kui; Sims, Harold F.; Jenkins, Christopher M.; Liu, Xinping; Mancuso, David J.; Zhao, Zhongdan; Guan, Shaoping; Abendschein, Dana R.; Han, Xianlin; Gross, Richard W.

    2012-01-01

    Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra-18:2 molecular species without a change in total cardiolipin content. Mechanistic analysis demonstrated that these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites, including a marked increase in the cardioprotective eicosanoid 14,15-epoxyeicosatrienoic acid. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency as evidenced by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic

  10. Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

    PubMed Central

    Eberhardt, S; Korn, S; Lottspeich, F; Bacher, A

    1997-01-01

    Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. PMID:9139911

  11. Pharmacological modulation of human platelet leukotriene C4-synthase.

    PubMed

    Sala, A; Folco, G; Henson, P M; Murphy, R C

    1997-03-21

    The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

  12. Ceramide Synthase-dependent Ceramide Generation and Programmed Cell Death

    PubMed Central

    Mullen, Thomas D.; Jenkins, Russell W.; Clarke, Christopher J.; Bielawski, Jacek; Hannun, Yusuf A.; Obeid, Lina M.

    2011-01-01

    The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death. PMID:21388949

  13. Type III polyketide synthases in microorganisms.

    PubMed

    Katsuyama, Yohei; Ohnishi, Yasuo

    2012-01-01

    Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which catalyze the condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions. Type III PKSs have been found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are involved in the biosynthesis of some lipidic compounds and various secondary metabolites, have several interesting characteristics that are not shared by plant type III PKSs. Further, many compounds produced by microbial type III PKSs have significant biological functions and/or important pharmaceutical activities. Thus, studies on this class of enzymes will expand our knowledge of the biosynthetic machineries that generate natural products and generate new findings about microbial physiology. The recent development of next-generation DNA sequencing has allowed for an increase in the number of microbial genomes sequenced and the discovery of many microbial type III PKS genes. Here, we describe basic methods to study microbial type III PKSs whose genes are easy to clone. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. 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.

  15. Structure of Leishmania major cysteine synthase

    PubMed Central

    Fyfe, Paul K.; Westrop, Gareth D.; Ramos, Tania; Müller, Sylke; Coombs, Graham H.; Hunter, William N.

    2012-01-01

    Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization. PMID:22750854

  16. Inverted stereocontrol of iridoid synthase in snapdragon

    PubMed Central

    Kries, Hajo; Kellner, Franziska; Kamileen, Mohamed Omar; O'Connor, Sarah E.

    2017-01-01

    The natural product class of iridoids, found in various species of flowering plants, harbors astonishing chemical complexity. The discovery of iridoid biosynthetic genes in the medicinal plant Catharanthus roseus has provided insight into the biosynthetic origins of this class of natural product. However, not all iridoids share the exact five- to six-bicyclic ring scaffold of the Catharanthus iridoids. For instance, iridoids in the ornamental flower snapdragon (Antirrhinum majus, Plantaginaceae family) are derived from the C7 epimer of this scaffold. Here we have cloned and characterized the iridoid synthase enzyme from A. majus (AmISY), the enzyme that is responsible for converting 8-oxogeranial into the bicyclic iridoid scaffold in a two-step reduction–cyclization sequence. Chiral analysis of the reaction products reveals that AmISY reduces C7 to generate the opposite stereoconfiguration in comparison with the Catharanthus homologue CrISY. The catalytic activity of AmISY thus explains the biosynthesis of 7-epi-iridoids in Antirrhinum and related genera. However, although the stereoselectivity of the reduction step catalyzed by AmISY is clear, in both AmISY and CrISY, the cyclization step produces a diastereomeric mixture. Although the reduction of 8-oxogeranial is clearly enzymatically catalyzed, the cyclization step appears to be subject to less stringent enzyme control. PMID:28701463

  17. Transmembrane topology of ceramide synthase in yeast

    PubMed Central

    Kageyama-Yahara, Natsuko; Riezman, Howard

    2006-01-01

    Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating cell-fate decisions. Three genes, LAG1, LAC1 and LIP1, have been shown to be required for ceramide synthase activity in Saccharomyces cerevisiae [Guillas, Kirchman, Chuard, Pfefferli, Jiang, Jazwinski and Conzelman (2001) EMBO J. 20, 2655–2665; Schorling, Vallee, Barz, Reizman and Oesterhelt (2001) Mol. Biol. Cell 12, 3417–3427; Vallee and Riezman (2005) EMBO J. 24, 730–741]. In the present study, the topology of the Lag1p and Lac1p subunits was investigated. The N- and C-termini of the proteins are in the cytoplasm and eight putative membrane-spanning domains were identified in Lag1p and Lac1p by insertion of glycosylation and factor Xa cleavage sites at various positions. The conserved Lag motif, potentially containing the active site, is most likely embedded in the membrane. We also present evidence that histidine and aspartic acid residues in the Lag motif are essential for the function of Lag1p in vivo. PMID:16756512

  18. 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

  19. Nitric Oxide Synthases in Heart Failure

    PubMed Central

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  20. The Eucalyptus terpene synthase gene family.

    PubMed

    Külheim, Carsten; Padovan, Amanda; Hefer, Charles; Krause, Sandra T; Köllner, Tobias G; Myburg, Alexander A; Degenhardt, Jörg; Foley, William J

    2015-06-11

    Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus. Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

  1. Electric Field Driven Torque in ATP Synthase

    PubMed Central

    Miller, John H.; Rajapakshe, Kimal I.; Infante, Hans L.; Claycomb, James R.

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring. PMID:24040370

  2. Electric field driven torque in ATP synthase.

    PubMed

    Miller, John H; Rajapakshe, Kimal I; Infante, Hans L; Claycomb, James R

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.

  3. Inducible nitric oxide synthase in the myocard.

    PubMed

    Buchwalow, I B; Schulze, W; Karczewski, P; Kostic, M M; Wallukat, G; Morwinski, R; Krause, E G; Müller, J; Paul, M; Slezak, J; Luft, F C; Haller, H

    2001-01-01

    Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

  4. Structures of human constitutive nitric oxide synthases

    PubMed Central

    Li, Huiying; Jamal, Joumana; Plaza, Carla; Pineda, Stephanie Hai; Chreifi, Georges; Jing, Qing; Cinelli, Maris A.; Silverman, Richard B.; Poulos, Thomas L.

    2014-01-01

    Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure–activity–relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme–inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03 Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73 Å resolution. PMID:25286850

  5. Nitric oxide synthase in the pineal gland.

    PubMed

    López-Figueroa, M O; Møller, M

    1996-10-01

    The recent discovery of nitric oxide (NO) as a biological messenger molecule with unique characteristics has opened a new field in pineal research. This free radical gas is synthesized by the enzyme nitric oxide synthase (NOS) from L-arginine. The activation of adrenoreceptors in the membrane of the pinealocytes mediates the increase in NO through a mechanism that involves G proteins. In the pinealocyte, NO stimulates guanylyl cyclase resulting in an increased intracellular content of cGMP. The role of cGMP in pineal metabolism, however, is still enigmatic. Using enzyme histochemistry and immunohistochemistry, the presence of NOS has been confirmed in the pineal gland of some species. In the rat and especially in the sheep, NOS is located in nerve fibres innervating the gland. These nerve fibres also contain the neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI), and are probably of parasympathetic origin. In cell cultures and tissue sections NOS immunoreactivity has been shown to be present in pinealocytes of the rat and bovine but not in the sheep. Finally, NOS is also present in the endothelial cells of the blood vessels of the pineal gland. Accordingly, in the mammalian pineal gland, NO is synthesized in both presynaptic nerve fibers and pinealocytes, as well as in blood vessels. However, the anatomical location of NO synthesis varies considerably among species. NO released in the pineal gland, might influence both the pineal metabolism and the blood flow of the gland.

  6. Anthranilate synthase subunit organization in Chromobacterium violaceum.

    PubMed

    Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

    2008-09-16

    Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

  7. Orientation-selective incorporation of transmembrane F0F1 ATP synthase complex from micrococcus luteus in polymer-supported membranes.

    PubMed

    Tutus, Murat; Rossetti, Fernanda F; Schneck, Emanuel; Fragneto, Giovanna; Förster, Friedrich; Richter, Ralf; Nawroth, Thomas; Tanaka, Motomu

    2008-11-10

    We report the vectorial incorporation of a highly asymmetric F0F1 ATP synthase complex from Micrococcus luteus into polymer-supported membranes. Dynamic light scattering and cryo electron microscopy confirm that the use of weak surfactants (bile acid) allows for the non-disruptive protein incorporation into lipid vesicles. Spreading of vesicles with ATP synthase onto a cellulose support results in a homogeneous distribution of proteins, in contrast to a patchy image observed on bare glass slides. The orientation of ATP synthase can be identified using an antibody to the ATP binding site as well as from topographic profiles of the surface. The method to "align" transmembrane proteins in supported membranes would open a possibility to quantify protein functions in biomimetic model systems.

  8. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    PubMed

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  9. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    PubMed Central

    Xu, Ting; Pagadala, Vijayakanth; Mueller, David M.

    2015-01-01

    The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs. PMID:25938092

  10. The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae.

    PubMed

    Choquer, Mathias; Dekkers, Katherine L; Chen, Hui-Qin; Cao, Lihua; Ueng, Peter P; Daub, Margaret E; Chung, Kuang-Ren

    2005-05-01

    Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.

  11. Effect of chronologic age on induction of cystathionine synthase, uroporphyrinogen I synthase, and glucose-6-phosphate dehydrogenase activities in lymphocytes.

    PubMed Central

    Gartler, S M; Hornung, S K; Motulsky, A G

    1981-01-01

    The activities of cystathionine synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8], and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) have been measured in phytohemagglutinin-stimulated lymphocytes of young and old human subjects. A significant decrease in activity with age was observed for cystathionine synthase and uroporphyrinogen I synthase but not for glucose-6-phosphate dehydrogenase. These changes could not be related to declining phytohemagglutinin response with aging. Age-related decreases in activity of some enzymes may be relevant for an understanding of the biology of aging. False assignment of heterozygosity, and even homozygosity, for certain genetic disorders, such as homocystinuria, may result when low enzyme levels are detected in the lymphocytes of older people. PMID:6940198

  12. Broadband Visible Light Induced NO Formation

    SciTech Connect

    Lubart, Rachel; Eichler, Maor; Friedmann, Harry; Ankri, Rinat; Savion, N.; Breitbart, Haim

    2009-06-19

    Nitric oxide formation is a potential mechanism for photobiomodulation because it is synthesized in cells by nitric oxide synthase (NOS), which contains both flavin and heme, and thus absorbs visible light. The purpose of this work was to study broadband visible light induced NO formation in various cells. Cardiac, endothelial, sperm cells and RAW 264.7 macrophages were illuminated with broadband visible light, 40-130 mW/cm2, 2.4-39 J/cm2, and nitric oxide production was quantified by using the Griess reagent. The results showed that visible light illumination increased NO concentration both in sperm and endothelial cells, but not in cardiac cells. Activation of RAW 264.7 macrophages was very small. It thus appears that NO is involved in photobiomodulation, though different light parameters and illumination protocols are needed to induce NO in various cells.

  13. The role of sphingomyelin and sphingomyelin synthases in cell death, proliferation and migration-from cell and animal models to human disorders.

    PubMed

    Taniguchi, Makoto; Okazaki, Toshiro

    2014-05-01

    Sphingomyelin constitutes membrane microdomains such as lipid raft, caveolae, and clathrin-coated pits and implicates in the regulation of trans-membrane signaling. On the other hand, sphingomyelin emerges as an important molecule to generate bioactive sphingolipids through ceramide. Sphingomyelin synthase is an enzyme that generates sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Although ceramide has a well-known role as a lipid mediator to regulate cell death and survival, the only known biological role of sphingomyelin regulated by sphingomyelin synthases was limited to being a source of bioactive lipids. Here, we describe the basic characters of sphingomyelin synthases and discuss additional roles for sphingomyelin and sphingomyelin synthase in biological functions including cell migration, apoptosis, autophagy, and cell survival/proliferation as well as in human disorders such as cancer and cardiovascular disorders. It is expected that a better understanding of the role of sphingomyelin regulated by sphingomyelin synthase will shed light on new mechanisms in cell biology, physiology and pathology. In the future, novel therapeutic procedures for currently incurable diseases could be developed through modifying the function of not only sphingolipids, such as sphingomyelin and ceramide, but also of their regulatory enzymes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

  14. Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase.

    PubMed

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H-J

    2012-05-25

    Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.

  15. Identification of a Dolabellane Type Diterpene Synthase and other Root-Expressed Diterpene Synthases in Arabidopsis

    PubMed Central

    Wang, Qiang; Jia, Meirong; Huh, Jung-Hyun; Muchlinski, Andrew; Peters, Reuben J.; Tholl, Dorothea

    2016-01-01

    Arabidopsis thaliana maintains a complex metabolism for the production of secondary or specialized metabolites. Such metabolites include volatile and semivolatile terpenes, which have been associated with direct and indirect defensive activities in flowers and leaves. In comparison, the structural diversity and function of terpenes in Arabidopsis roots has remained largely unexplored despite a substantial number of root-expressed genes in the Arabidopsis terpene synthase (TPS) gene family. We show that five root-expressed TPSs of an expanded subfamily-a type clade in the Arabidopsis TPS family function as class I diterpene synthases that predominantly convert geranylgeranyl diphosphate (GGPP) to different semi-volatile diterpene products, which are in part detectable at low levels in the ecotypes Columbia (Col) and Cape Verde Island (Cvi). The enzyme TPS20 produces a macrocyclic dolabellane diterpene alcohol and a dolabellane-related diterpene olefin named dolathaliatriene with a so far unknown C6-C11 bicyclic scaffold besides several minor olefin products. The TPS20 compounds occur in all tissues of Cvi but are absent in the Col ecotype because of deletion and substitution mutations in the Col TPS20 sequence. The primary TPS20 diterpene products retard the growth of the root rot pathogen Pythium irregulare but only at concentrations exceeding those in planta. Together, our results demonstrate that divergence and pseudogenization in the Arabidopsis TPS gene family allow for structural plasticity in diterpene profiles of above- and belowground tissues. PMID:27933080

  16. Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii.

    PubMed

    Zhao, Huan; Tang, Qi; Mo, Changming; Bai, Longhua; Tu, Dongping; Ma, Xiaojun

    2017-03-01

    Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS) and cycloartenol synthase (CAS) are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR) approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF) encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

  17. The rice ent-KAURENE SYNTHASE LIKE 2 encodes a functional ent-beyerene synthase.

    PubMed

    Tezuka, Daisuke; Ito, Akira; Mitsuhashi, Wataru; Toyomasu, Tomonobu; Imai, Ryozo

    2015-05-08

    The rice genome contains a family of kaurene synthase-like (OsKSL) genes that are responsible for the biosynthesis of various diterpenoids, including gibberellins and phytoalexins. While many OsKSL genes have been functionally characterized, the functionality of OsKSL2 is still unclear and it has been proposed to be a pseudogene. Here, we found that OsKSL2 is drastically induced in roots by methyl jasmonate treatment and we successfully isolated a full-length cDNA for OsKSL2. Sequence analysis of the OsKSL2 cDNA revealed that the open reading frame of OsKSL2 is mispredicted in the two major rice genome databases, IRGSP-RAP and MSU-RGAP. In vitro conversion assay indicated that recombinant OsKSL2 catalyzes the cyclization of ent-CDP into ent-beyerene as a major and ent-kaurene as a minor product. ent-Beyerene is an antimicrobial compound and OsKSL2 is induced by methyl jasmonate; these data suggest that OsKSL2 is a functional ent-beyerene synthase that is involved in defense mechanisms in rice roots.

  18. Functional analysis of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in sugarcane (Saccharum) cultivars.

    PubMed

    Verma, A K; Upadhyay, S K; Verma, P C; Solomon, S; Singh, S B

    2011-03-01

    Sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (SS; EC 2.4.1.13) are key enzymes in the synthesis and breakdown of sucrose in sugarcane. The activities of internodal SPS and SS, as well as transcript expression were determined using semi-quantitative RT-PCR at different developmental stages of high and low sucrose accumulating sugarcane cultivars. SPS activity and transcript expression was higher in mature internodes compared with immature internodes in all the studied cultivars. However, high sugar cultivars showed increased transcript expression and enzyme activity of SPS compared to low sugar cultivars at all developmental stages. SS activity was higher in immature internodes than in mature internodes in all cultivars; SS transcript expression showed a similar pattern. Our studies demonstrate that SPS activity was positively correlated with sucrose and negatively correlated with hexose sugars. However, SS activity was negatively correlated with sucrose and positively correlated with hexose sugars. The present study opens the possibility for improvement of sugarcane cultivars by increasing expression of the respective enzymes using transgene technology. © 2010 German Botanical Society and The Royal Botanical Society of the Netherlands.

  19. Light Visor

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Seasonal Affective Disorder is a form of depression brought on by reduced light. For some people, this can lead to clinical depression. NASA has conducted research in light therapy and employs it to help astronauts adjust internal rhythms during orbital flight. Dr. George Brainard, a medical researcher and NASA consultant, has developed a portable light therapy device, which is commercially available. The Light Visor allows continuous light therapy and can be powered by either batteries or electricity. Dr. Brainard continues to research various aspects of light therapy.

  20. Nitric oxide synthase in cardiac sarcoplasmic reticulum.

    PubMed

    Xu, K Y; Huso, D L; Dawson, T M; Bredt, D S; Becker, L C

    1999-01-19

    NO. is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO. produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of L-arginine to L-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO. was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOS mu antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO. produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart.

  1. Nitric oxide synthase in cardiac sarcoplasmic reticulum

    PubMed Central

    Xu, Kai Y.; Huso, David L.; Dawson, Ted M.; Bredt, David S.; Becker, Lewis C.

    1999-01-01

    NO⋅ is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO⋅ produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of l-arginine to l-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO⋅ was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOSμ antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO⋅ produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart. PMID:9892689

  2. Tertiary model of a plant cellulose synthase

    PubMed Central

    Sethaphong, Latsavongsakda; Haigler, Candace H.; Kubicki, James D.; Zimmer, Jochen; Bonetta, Dario; DeBolt, Seth; Yingling, Yaroslava G.

    2013-01-01

    A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose–synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs. PMID:23592721

  3. Allosteric inhibition of human porphobilinogen synthase.

    PubMed

    Lawrence, Sarah H; Ramirez, Ursula D; Selwood, Trevor; Stith, Linda; Jaffe, Eileen K

    2009-12-18

    Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

  4. Allosteric Inhibition of Human Porphobilinogen Synthase*

    PubMed Central

    Lawrence, Sarah H.; Ramirez, Ursula D.; Selwood, Trevor; Stith, Linda; Jaffe, Eileen K.

    2009-01-01

    Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329–337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ∼111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90–100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme. PMID:19812033

  5. Nitric oxide synthases: structure, function and inhibition.

    PubMed Central

    Alderton, W K; Cooper, C E; Knowles, R G

    2001-01-01

    This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated. PMID:11463332

  6. Modulation of ceramide synthase activity via dimerization.

    PubMed

    Laviad, Elad L; Kelly, Samuel; Merrill, Alfred H; Futerman, Anthony H

    2012-06-15

    Ceramide, the backbone of all sphingolipids, is synthesized by a family of ceramide synthases (CerS) that each use acyl-CoAs of defined chain length for N-acylation of the sphingoid long chain base. CerS mRNA expression and enzymatic activity do not always correlate with the sphingolipid acyl chain composition of a particular tissue, suggesting post-translational mechanism(s) of regulation of CerS activity. We now demonstrate that CerS activity can be modulated by dimer formation. Under suitable conditions, high M(r) CerS complexes can be detected by Western blotting, and various CerS co-immunoprecipitate. CerS5 activity is inhibited in a dominant-negative fashion by co-expression with catalytically inactive CerS5, and CerS2 activity is enhanced by co-expression with a catalytically active form of CerS5 or CerS6. In a constitutive heterodimer comprising CerS5 and CerS2, the activity of CerS2 depends on the catalytic activity of CerS5. Finally, CerS dimers are formed upon rapid stimulation of ceramide synthesis by curcumin. Together, these data demonstrate that ceramide synthesis can be regulated by the formation of CerS dimers and suggest a novel way to generate the acyl chain composition of ceramide (and downstream sphingolipids), which may depend on the interaction of CerS with each other.

  7. In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase.

    PubMed

    Peters, Verena; Rehm, Bernd H A

    2006-03-01

    This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The beta-galactosidase was fused to the N terminus of the class II PHA synthase from Pseudomonas aeruginosa. The open reading frame was confirmed to encode the complete fusion protein by T7 promoter-dependent overexpression. Restoration of PHA biosynthesis in the PHA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase function of the fusion protein. PHA granules were isolated and showed beta-galactosidase activity. PHA granule attached proteins were analyzed and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Surprisingly, the beta-galactosidase-PHA synthase fusion protein was detectable at a high copy number at the PHA granule, compared with PHA synthase alone, which was barely detectable at PHA granules. Localization of the beta-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-beta-galactosidase antibodies. Treatment of these beta-galactosidase-PHA granules with urea suggested a covalent binding of the beta-galactosidase-PHA synthase to the PHA granule. The immobilized beta-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A Km of 630 microM and a Vmax of 17.6 nmol/min for orthonitrophenyl-beta-D-galactopyranoside as a substrate was obtained. The immobilized beta-galactosidase was stable for at least several months under various storage conditions. This study demonstrated that protein engineering of PHA synthase enables the manufacture of PHA granules with covalently attached enzymes, suggesting an application in recycling of biocatalysts, such as in fine-chemical production.

  8. 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

  9. Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase

    SciTech Connect

    Cook, Paul D.; Carney, Amanda E.; Holden, Hazel M.

    2009-01-12

    Perosamine (4-amino-4,6-dideoxy-d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 {angstrom} resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub cat} was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy-d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

  10. Light Motives.

    ERIC Educational Resources Information Center

    Filler, Martin

    1979-01-01

    The new energy consciousness has led to a thorough reevaluation of how artificial lighting can be used wisely, while other researchers have explored the potential of daylighting as an alternative interior light source. (Author/MLF)

  11. Light Reflector

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Ultra Sales, Inc.'s fluorescent lighting fixture gets a boost in reflectivity through installation of Lightdriver, a thin tough thermoplastic film plated with aluminum, capable of reflecting 95 percent of visible light striking it. Lightdriver increases brightness without adding bulbs, and allows energy savings by removing some bulbs because the mirrorlike surface cuts light loss generally occasioned by conventional low reflectivity white painted surface above the bulbs in many fluorescent fixtures. Forty-five percent reduction in lighting electricity is attainable.

  12. Lighting Utilization.

    ERIC Educational Resources Information Center

    Crank, Ron

    This instructional unit is one of 10 developed by students on various energy-related areas that deals specifically with lighting utilization. Its objective is for the student to be able to outline the development of lighting use and conservation and identify major types and operating characteristics of lamps used in electric lighting. Some topics…

  13. SCHOOL LIGHTING

    ERIC Educational Resources Information Center

    1965

    SEVERAL ARTICLES ON SCHOOL LIGHTING ARE CONTAINED IN THIS JOURNAL. THE TITLES AND AUTHORS INCLUDED ARE AS FOLLOWS--(1) "TODAY'S CONCEPTS IN SCHOOL LIGHTING" BY CHARLES D. GIBSON, (2) "CHALLENGE OF TOMMORROW'S LIGHTING" BY S.K. GUTH AND E.H. WITTE, (3) "PEEK PREVIEW OF THE WINDOWLESS SCHOOL" BY JAMES J. MORISSEAU, (4) "MAINTENANCE BEGINS BEFORE…

  14. SCHOOL LIGHTING

    ERIC Educational Resources Information Center

    1965

    SEVERAL ARTICLES ON SCHOOL LIGHTING ARE CONTAINED IN THIS JOURNAL. THE TITLES AND AUTHORS INCLUDED ARE AS FOLLOWS--(1) "TODAY'S CONCEPTS IN SCHOOL LIGHTING" BY CHARLES D. GIBSON, (2) "CHALLENGE OF TOMMORROW'S LIGHTING" BY S.K. GUTH AND E.H. WITTE, (3) "PEEK PREVIEW OF THE WINDOWLESS SCHOOL" BY JAMES J. MORISSEAU, (4) "MAINTENANCE BEGINS BEFORE…

  15. Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

    PubMed Central

    Wong, H C; Fear, A L; Calhoon, R D; Eichinger, G H; Mayer, R; Amikam, D; Benziman, M; Gelfand, D H; Meade, J H; Emerick, A W

    1990-01-01

    An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum. Images PMID:2146681

  16. Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

    PubMed

    Roach, Christopher R; Hall, Dawn E; Zerbe, Philipp; Bohlmann, Jörg

    2014-08-22

    The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

    PubMed Central

    Eldar-Finkelman, H; Argast, G M; Foord, O; Fischer, E H; Krebs, E G

    1996-01-01

    In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8816781

  18. ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry

    PubMed Central

    Hoffmann, Jan; Sokolova, Lucie; Preiss, Laura; Hicks, David B.; Krulwich, Terry A.; Morgner, Nina; Wittig, Ilka; Schägger, Hermann; Meier, Thomas; Brutschy, Bernd

    2010-01-01

    Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F1Fo-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F1Fo-ATP synthases. Under selected buffer conditions the mass of the intact F1Fo-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases. PMID:20820587

  19. Insulin stimulation of glycogen synthase in cultured human diploid fibroblasts.

    PubMed

    Hidaka, H; Howard, B V; Kosmakos, F C; Fields, R M; Craig, J W; Bennett, P H; Larner, J

    1980-10-01

    The effect of insulin on glycogen synthase activity in human diploid fibroblasts has been studied. As little as 2 X 10(-10) M insulin increased the glycogen synthase / activity without changing the total activity. Stimulation occurred within 5 min and became maximal in 30 min. A half-maximal increase of / activity was achieved at 3 X 10(-9) M insulin. Glucose starvation increased the magnitude of response of glycogen synthase to insulin but did not change the insulin concentration necessary to give a half-maximal stimulation. Glucose increased the basal level of / activity in human diploid fibroblasts; the effect of insulin was additive. During in vitro senescence the total glycogen synthase activity declined, but the concentration of insulin that produced a half-maximal stimulation remained unchanged. These data indicate that regulation of glycogen synthase activity in human diploid fibroblasts is responsive to physiologic insulin levels and that the system provides a useful model for the in vitro study of insulin sensitivity.

  20. Saccharomyces cerevisiae contains two functional citrate synthase genes.

    PubMed Central

    Kim, K S; Rosenkrantz, M S; Guarente, L

    1986-01-01

    The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed. Images PMID:3023912

  1. Heterologous expression of an active chitin synthase from Rhizopus oryzae.

    PubMed

    Salgado-Lugo, Holjes; Sánchez-Arreguín, Alejandro; Ruiz-Herrera, José

    2016-12-01

    Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.

  2. Identification of two distinct Bacillus subtilis citrate synthase genes.

    PubMed

    Jin, S; Sonenshein, A L

    1994-08-01

    Two distinct Bacillus subtilis genes (citA and citZ) were found to encode citrate synthase isozymes that catalyze the first step of the Krebs cycle. The citA gene was cloned by genetic complementation of an Escherichia coli citrate synthase mutant strain (W620) and was in a monocistronic transcriptional unit. A divergently transcribed gene, citR, could encode a protein with strong similarity to the bacterial LysR family of regulatory proteins. A null mutation in citA had little effect on citrate synthase enzyme activity or sporulation. The residual citrate synthase activity was purified from a citA null mutant strain, and the partial amino acid sequence for the purified protein (CitZ) was determined. The citZ gene was cloned from B. subtilis chromosomal DNA by using a PCR-generated probe synthesized with oligonucleotide primers derived from the partial amino acid sequence of purified CitZ. The citZ gene proved to be the first gene in a tricistronic cluster that also included citC (coding for isocitrate dehydrogenase) and citH (coding for malate dehydrogenase). A mutation in citZ caused a substantial loss of citrate synthase enzyme activity, glutamate auxotrophy, and a defect in sporulation.

  3. Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus

    PubMed Central

    Agger, Sean; Lopez-Gallego, Fernando; Schmidt-Dannert, Claudia

    2009-01-01

    SUMMARY Fungi are a rich source of bioactive secondary metabolites and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared to the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as a α-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes δ-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homolog, but instead was found to catalyze highly specific the synthesis of α-cuprenene. Co-expression of cop6 and the two monooxygenase genes next to it yields oxygenated α-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species. PMID:19400802

  4. Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus.

    PubMed

    Agger, Sean; Lopez-Gallego, Fernando; Schmidt-Dannert, Claudia

    2009-06-01

    Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an alpha-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes delta-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of alpha-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated alpha-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.

  5. Light Source

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Research on food growth for long duration spacecraft has resulted in a light source for growing plants indoors known as Qbeam, a solid state light source consisting of a control unit and lamp. The light source, manufactured by Quantum Devices, Inc., is not very hot, although it generates high intensity radiation. When Ron Ignatius, an industrial partner of WCSAR, realized that terrestrial plant research lighting was not energy efficient enough for space use, he and WCSAR began to experiment with light emitting diodes. A line of LED products was developed, and QDI was formed to market the technology. An LED-based cancer treatment device is currently under development.

  6. The Crystal Structures of the Open and Catalytically Competent Closed Conformation of Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Jia, Xiaofei; Yep, Alejandra; Preiss, Jack; Geiger, James H.

    2009-07-06

    Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site

  7. In Vivo and in Vitro Synthesis of Phosphatidylglycerol by an Escherichia coli Cardiolipin Synthase.

    PubMed

    Li, Chijun; Tan, Brandon K; Zhao, Jinshi; Guan, Ziqiang

    2016-11-25

    Phosphatidylglycerol (PG) makes up 5-20% of the phospholipids of Escherichia coli and is essential for growth in wild-type cells. PG is synthesized from the dephosphorylation of its immediate precursor, phosphatidylglycerol phosphate (PGP) whose synthase in E. coli is PgsA. Using genetic, biochemical, and highly sensitive mass spectrometric approaches, we identified an alternative mechanism for PG synthesis in E. coli that is PgsA independent. The reaction of synthesis involves the conversion of phosphatidylethanolamine and glycerol into PG and is catalyzed by ClsB, a phospholipase D-type cardiolipin synthase. This enzymatic reaction is demonstrated herein both in vivo and in vitro as well as by using the purified ClsB protein. When the growth medium was supplemented with glycerol, the expression of E. coli ClsB significantly increased PG and cardiolipin levels, with the growth deficiency of pgsA null strain also being complemented under such conditions. Identification of this alternative mechanism for PG synthesis not only expands our knowledge of bacterial anionic phospholipid biosynthesis, but also sheds light on the biochemical functions of the cls gene redundancy in E. coli and other bacteria. Finally, the PGP-independent PG synthesis in E. coli may also have important implications for the understanding of PG biosynthesis in eukaryotes that remains incomplete. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Methionine synthase and thymidylate synthase gene polymorphisms and colorectal adenoma risk: the self defense forces study.

    PubMed

    Yoshimitsu, Shinichiro; Morita, Makiko; Hamachi, Tadamichi; Tabata, Shinji; Abe, Hiroshi; Tajima, Osamu; Uezono, Kousaku; Ohnaka, Keizo; Kono, Suminori

    2012-10-01

    Folate-mediated one-carbon metabolism has been implicated in colorectal carcinogenesis. We investigated associations of functional genetic polymorphisms of methionine synthase (MTR), MTR reductase (MTRR), and thymidylate synthase (TS) with colorectal adenomas. The study subjects were 455 cases of colorectal adenomas and 1052 controls with no polyp at colonoscopy. Genotypes were determined for MTR A2756G, MTRR A66G and two polymorphisms in the TS gene, 28-bp tandem repeat polymorphism in the promoter enhancer region (TSER) and 6-bp deletion polymorphism at position 1494 in the 3' untranslated region (TS 1494del6). We also examined the alcohol-genotype and gene-gene interactions on adenoma risk. The GG genotype of MTR A2756G was associated with an increased risk of colorectal adenomas; odds ratios for AG and GG versus AA genotype were 0.99 (95% confidence interval 0.78-1.26) and 1.72 (1.04-2.82), respectively. The increase in the risk associated with MTR 2756GG genotype was evident in men with high alcohol consumption (≥30 mL/d), but not in those with low alcohol consumption (interaction P = 0.03). Men who were homozygous for the TSER double-repeat allele had a slightly decreased risk of colorectal adenomas as compared with those homozygous for the TSER triple-repeat allele. Neither MTRR A66G nor TS 1494del6 was associated with colorectal adenomas. There was no measurable interaction either between MTR A2756G and MTRR A66G or between TSER and TS 1494del6. MTR A2756G appears to be associated with colorectal adenoma risk differently according to alcohol consumption. The MTR-catalyzed reaction may play an important role in the development of colorectal adenomas.

  9. 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.

  10. Nucleotide sequence of the chicken 5-aminolevulinate synthase gene.

    PubMed Central

    Maguire, D J; Day, A R; Borthwick, I A; Srivastava, G; Wigley, P L; May, B K; Elliott, W H

    1986-01-01

    5-Aminolevulinate synthase, the first and rate-controlling enzyme of heme biosynthesis, is regulated in the liver by the end-product heme. To study this negative control mechanism, we have isolated the chicken gene for ALA-synthase and determined the nucleotide sequence. The structural gene is 6.9 kb long and contains 10 exons. The transcriptional start site for ALA-synthase was determined by primer extension analysis. A fragment of 291 bp from the 5' flanking region including 34 bp of the first exon shows promoter activity when introduced upstream of a chicken histone H2B gene and injected into the nuclei of Xenopus laevis oocytes. Images PMID:3005973

  11. Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria.

    PubMed

    Whitney, J C; Howell, P L

    2013-02-01

    The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an inner-membrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane β-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-β-D-N-acetylglucosamine (PNAG) secretion systems. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria

    PubMed Central

    Whitney, J.C.; Howell, P.L.

    2014-01-01

    The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an inner-membrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane β-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-β-D-N-acetylglucosamine (PNAG) secretion systems. PMID:23117123

  13. Properties of peroxisomal and mitochondrial citrate synthase from Agave americana.

    PubMed

    Segovia, J L; Zafra, M F; Alejandre, M J; García-Peregrín, E

    1982-09-01

    Adenine nucleotides were tested as effectors of peroxisomal and mitochondrial citrate synthase from Agave americana leaves in the presence of different concentrations of acetyl-CoA and oxalacetate substrates. ATP inhibited both enzyme activities but with a different inhibition profile. 1.0-7.5 mM ADP did not inhibit the peroxisomal citrate synthase in the presence of high substrate concentrations, while the mitochondrial enzyme was strongly inhibited by 1.0 mM ADP in the same conditions. Likewise, a different pattern was obtained with AMP on both peroxisomal and mitochondrial activities. The rate of citrate formation as function of acetyl-CoA and oxalacetate concentration was also studied in both fractions. Maximal velocity was highest in the peroxisomal fraction, whether acetyl-CoA or oxalacetate were the variable substrates. These differences indicate that peroxisomal and mitochondrial citrate synthases seem to be two different isoenzymes.

  14. The hyaluronate synthase from a eukaryotic cell line.

    PubMed Central

    Klewes, L; Turley, E A; Prehm, P

    1993-01-01

    The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 7 PMID:8457208

  15. Directed evolution of squalene synthase for dehydrosqualene biosynthesis.

    PubMed

    Furubayashi, Maiko; Li, Ling; Katabami, Akinori; Saito, Kyoichi; Umeno, Daisuke

    2014-09-17

    Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C30 carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a "dehydrosqualene synthase." All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Kinetic characteristics of nitric oxide synthase from rat brain.

    PubMed Central

    Knowles, R G; Palacios, M; Palmer, R M; Moncada, S

    1990-01-01

    The relationship between the rate of synthesis of nitric oxide (NO) and guanylate cyclase stimulation was used to characterize the kinetics of the NO synthase from rat forebrain and of some inhibitors of this enzyme. The NO synthase had an absolute requirement for L-arginine and NADPH and did not require any other cofactors. The enzyme had a Vmax. of 42 pmol of NO formed.min-1.mg of protein-1 and a Km for L-arginine of 8.4 microM. Three analogues of L-arginine, namely NG-monomethyl-L-arginine, NG-nitro-L-arginine and NG-iminoethyl-L-ornithine inhibited the brain NO synthase. All three compounds were competitive inhibitors of the enzyme with Ki values of 0.7, 0.4 and 1.2 microM respectively. PMID:1695842

  17. Utility of Aspergillus niger citrate synthase promoter for heterologous expression.

    PubMed

    Dave, Kashyap; Punekar, Narayan S

    2011-09-10

    Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi.

  18. Evolutionary and functional analysis of mulberry type III polyketide synthases.

    PubMed

    Li, Han; Liang, Jiubo; Chen, Hu; Ding, Guangyu; Ma, Bi; He, Ningjia

    2016-08-04

    Type III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses. Eighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol. Characterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants.

  19. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    PubMed

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  20. Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A

    PubMed Central

    Choi, Hyeon-Son; Han, Gil-Soo; Carman, George M.

    2010-01-01

    The CHO1-encoded phosphatidylserine synthase from Saccharomyces cerevisiae is phosphorylated and inhibited by protein kinase A in vitro. CHO1 alleles bearing Ser46 → Ala and/or Ser47 → Ala mutations were constructed and expressed in a cho1Δ mutant lacking phosphatidylserine synthase. In vitro, the S46A/S47A mutation reduced the total amount of phosphorylation by 90% and abolished the inhibitory effect protein kinase A had on phosphatidylserine synthase activity. The enzyme phosphorylation by protein kinase A, which was time- and dose-dependent and dependent on the concentration of ATP, caused a electrophoretic mobility shift from a 27-kDa form to a 30-kDa form. The two electrophoretic forms of phosphatidylserine synthase were present in exponential phase cells, whereas only the 27-kDa form was present in stationary phase cells. In vivo labeling with 32Pi and immune complex analysis showed that the 30-kDa form was predominantly phosphorylated when compared with the 27-kDa form. However, the S46A/S47A mutations abolished the protein kinase A-mediated electrophoretic mobility shift. The S46A/S47A mutations also caused a 55% reduction in the total amount of phosphatidylserine synthase in exponential phase cells and a 66% reduction in the amount of enzyme in stationary phase cells. In phospholipid composition analysis, cells expressing the S46A/S47A mutant enzyme exhibited a 57% decrease in phosphatidylserine and a 40% increase in phosphatidylinositol. These results indicate that phosphatidylserine synthase is phosphorylated on Ser46 and Ser47 by protein kinase A, which results in a higher amount of enzyme for the net effect of stimulating the synthesis of phosphatidylserine. PMID:20145252

  1. Mapping a kingdom-specific functional domain of squalene synthase.

    PubMed

    Linscott, Kristin B; Niehaus, Thomas D; Zhuang, Xun; Bell, Stephen A; Chappell, Joe

    2016-09-01

    Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Allosteric regulation of glycogen synthase in liver. A physiological dilemma.

    PubMed

    Nuttall, F Q; Gannon, M C

    1993-06-25

    Glycogen synthase catalyzes the transfer of the glucosyl moiety from UDP-glucose to the terminal branch of the glycogen molecule and is considered to be the rate-limiting enzyme for glycogen synthesis. However, under ideal assay conditions, i.e. 37 degrees C with saturating concentrations of UDP-glucose and the activator, glucose-6-P, the maximal catalytic activity of glycogen synthase was only 78% of the in vivo glycogen synthetic rate. Using concentrations of UDP-glucose and glucose-6-P likely to be present in vivo, the rate was only approximately 30%. This prompted us to reassess a possible role of allosteric effectors on synthase activity. Glycogen synthase was assayed at 37 degrees C using dilute, pH 7.0, buffered extracts, initial rate conditions, and UDP-glucose and glucose-6-P concentrations, which approximate those calculated to be present in total liver cell water. Several allosteric effectors were tested. Magnesium and AMP had little effect on activity. Pi, ADP, ATP, and UTP inhibited activity. When a combination of effectors were added at concentrations approximating those present in cell water, synthase activity could account for only 2% of the glycogen synthetic rate. Thus, although allosteric effectors are likely to be playing a major role in regulating synthase enzymic activity in liver cells, to date, a metabolite that can stimulate activity and/or overcome nucleotide inhibition has yet to be identified. If such a metabolite cannot be identified, an additional or alternative pathway for glycogen synthesis must be considered.

  3. Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting.

    PubMed

    Ito, Junta; Uchida, Hiroyuki; Machida, Naomi; Ohtake, Kazuo; Saito, Yuki; Kobayashi, Jun

    2017-04-01

    promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation and apoptosis in crypts and villi. These novel findings elucidate the relationship between these NOS isoforms and its impact on recovery from intestinal injury. A mechanism is proposed comprising the up-regulation of nNOS activity by mechanical stimulation due to the presence of food in the intestine, restricting iNOS-associated apoptosis and promoting cell proliferation and gut motility. Our investigation sheds light on the molecular basis behind the repercussions of total parenteral nutrition on intestinal mucosal integrity, and more importantly, the beneficial effects of early enteral feeding.

  4. Inhibitors of Fatty Acid Synthase for Prostate Cancer

    DTIC Science & Technology

    2010-05-31

    targeting. Ursolic acid , a pentacyclic triterpenoid acid , as well as the tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate...2007,  6(7), 2120‐2126.  73.  Liu, Y., Tian, W., Ma, X., and Ding, W. Evaluation of  inhibition of  fatty  acid  synthase by  ursolic   acid : positive...AD_________________ Award Number: W81XWH-09-1-0204 TITLE: Inhibitors of Fatty Acid Synthase for

  5. Engineered biosynthesis of plant polyketides: manipulation of chalcone synthase.

    PubMed

    Abe, Ikuro; Watanabe, Tatsuya; Morita, Hiroyuki; Kohno, Toshiyuki; Noguchi, Hiroshi

    2006-02-02

    [reaction: see text]. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase catalyzing condensation of 4-coumaroyl-CoA with three molecules of malonyl-CoA. Surprisingly, it was demonstrated that S338V mutant of Scutellaria baicalensis CHS produced octaketides SEK4/SEK4b from eight molecules of malonyl-CoA. Further, the octaketides-forming activity was dramatically increased in a CHS triple mutant (T197G/G256L/S338T). The functional conversion is based on the simple steric modulation of a chemically inert residue lining the active-site cavity.

  6. Molecular aspects of beta-ketoacyl synthase (KAS) catalysis.

    PubMed

    von Wettstein-Knowles, P; Olsen, J; Arnvig Mcguire, K; Larsen, S

    2000-12-01

    Crystal structure data for Escherichia coli beta-ketoacyl synthase (KAS) I with C(10) and C(12) fatty acid substrates bound in conjunction with results from mutagenizing residues in the active site leads to a model for catalysis. Differences from and similarities to the other Claisen enzymes carrying out decarboxylations reveal two catalytic mechanisms, one for KAS I and KAS II, the other for KAS III and chalcone synthase. A comparison of the structures of KAS I and KAS II does not reveal the basis of chain-length specificity. The structures of the Arabidopsis thaliana KAS family are compared.

  7. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  8. Brucella spp. lumazine synthase: a novel antigen delivery system.

    PubMed

    Sciutto, Edda; Toledo, Andrea; Cruz, Carmen; Rosas, Gabriela; Meneses, Gabriela; Laplagne, Diego; Ainciart, Natalia; Cervantes, Jacquelynne; Fragoso, Gladis; Goldbaum, Fernando A

    2005-04-15

    Lumazine synthase from Brucella spp. (BLS) was evaluated as a protein carrier to improve antigen delivery of KETc1, one of the peptides of the anti-cysticercosis vaccine. KETc1 becomes antigenic, preserved its immunogenicity and its protective capacity when expressed as a recombinant chimeric protein using Brucella spp. lumazine synthase. KETc1 and BLS-KETc1 were not MHC H-2(d), H-2(k) nor H-2(b) haplotype-restricted albeit KETc1 is preferentially presented in the H-2(b) haplotype. These findings support that BLS is a potent new delivery system for the improvement of subunit vaccines.

  9. Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.).

    PubMed

    Zeh, Michaela; Leggewie, Georg; Hoefgen, Rainer; Hesse, Holger

    2002-02-01

    A potato cDNA clone, StMS1, that encodes a methionine synthase was isolated. This protein was identified on the basis of both structural and functional evidence. The predicted sequence of the protein encoded by StMS1 shows a high degree of similarity to methionine synthases from other organisms and the expression of StMS1 in bacterial mutant strains restored the mutant's ability to synthesize methionine. Genomic organization and expression analyses suggest that StMS1 is a low-copy gene and is differentially expressed in potato organs. StMS1 expression was found in all tissues, but at elevated levels in flowers, basal levels in sink and source leaves, roots and stolons, and low levels in stems and tubers. RNA expression data were confirmed by western blot analysis except that the protein content in leaves was less than expected from the RNA data. Western blot analysis of subcellular fractions revealed that the protein is located in the cytosol. However, the changing pattern of gene expression during the day/night period implied a light-dependent control of MS transcription normally seen for enzymes localized in plastids. The expression of MS was shown to be light-inducible with its highest expression at midday. These RNA data were not confirmed at the protein level since protein content levels remained constant over the whole day. Feeding experiments of detached leaves revealed that sucrose or sucrose-derived products are responsible for StMS1 induction. This induction can be blocked by treatment with DCMU during the light period. Western analysis revealed that the amount of StMS1 is not affected by either treatment. This experiment confirmed the presence of a day/night rhythm. Methionine synthase expression is regulated by photoassimilates but this seems not to detectably alter protein levels.

  10. The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta[W

    PubMed Central

    Orlova, Irina; Nagegowda, Dinesh A.; Kish, Christine M.; Gutensohn, Michael; Maeda, Hiroshi; Varbanova, Marina; Fridman, Eyal; Yamaguchi, Shinjiro; Hanada, Atsushi; Kamiya, Yuji; Krichevsky, Alexander; Citovsky, Vitaly; Pichersky, Eran; Dudareva, Natalia

    2009-01-01

    Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. PMID:20028839

  11. New Mutations in the Mycobacterial ATP Synthase: New Insights into the Binding of the Diarylquinoline TMC207 to the ATP Synthase C-Ring Structure

    PubMed Central

    Segala, Elena; Sougakoff, Wladimir; Nevejans-Chauffour, Aurelie; Jarlier, Vincent

    2012-01-01

    TMC207 is a new antituberculous drug belonging to the diarylquinoline class which very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis, one of the most important pathogens in the world. In order to map the amino acid residues involved in the binding of the drug, we have selected in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria. Six distinct mutations, Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met, have been identified in the subunit c forming a C ring in the ATP synthase. They were studied by evaluating the levels of resistance that they confer in the selected clones and by using an isogenic complementation system in Mycobacterium smegmatis. The rates of increase of TMC207 MIC values (8- to 133-fold) were interpreted by constructing by homology modeling a structure of the mycobacterial C ring which was used for docking simulations with TMC207. Our results suggest that the residues found to be mutated in the resistant clones, together with a tyrosine specifically conserved at position 64 in mycobacteria, define a cleft located between two adjacent c subunits in the C ring. This cleft, which encompasses the proton-binding site (Glu61), is well fitted to bind TMC207 at the level of the bromoquinoline moiety, with the drug being anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28, respectively. These data shed light on the molecular interactions allowing TMC207 to bind specifically and efficiently at the level of the proton-binding site of the mycobacterial C ring. PMID:22354303

  12. New mutations in the mycobacterial ATP synthase: new insights into the binding of the diarylquinoline TMC207 to the ATP synthase C-ring structure.

    PubMed

    Segala, Elena; Sougakoff, Wladimir; Nevejans-Chauffour, Aurelie; Jarlier, Vincent; Petrella, Stephanie

    2012-05-01

    TMC207 is a new antituberculous drug belonging to the diarylquinoline class which very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis, one of the most important pathogens in the world. In order to map the amino acid residues involved in the binding of the drug, we have selected in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria. Six distinct mutations, Asp28 → Gly, Asp28 → Ala, Leu59 → Val, Glu61 → Asp, Ala63 → Pro, and Ile66 → Met, have been identified in the subunit c forming a C ring in the ATP synthase. They were studied by evaluating the levels of resistance that they confer in the selected clones and by using an isogenic complementation system in Mycobacterium smegmatis. The rates of increase of TMC207 MIC values (8- to 133-fold) were interpreted by constructing by homology modeling a structure of the mycobacterial C ring which was used for docking simulations with TMC207. Our results suggest that the residues found to be mutated in the resistant clones, together with a tyrosine specifically conserved at position 64 in mycobacteria, define a cleft located between two adjacent c subunits in the C ring. This cleft, which encompasses the proton-binding site (Glu61), is well fitted to bind TMC207 at the level of the bromoquinoline moiety, with the drug being anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28, respectively. These data shed light on the molecular interactions allowing TMC207 to bind specifically and efficiently at the level of the proton-binding site of the mycobacterial C ring.

  13. Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium.

    PubMed Central

    Nakamura, T; Iwahashi, H; Eguchi, Y

    1984-01-01

    S-Sulfocysteine synthase was isolated from Salmonella typhimurium LT-2 to homogeneous form with polyacrylamide gel electrophoresis. The molecular weight of this enzyme was determined to be ca. 55,000. The enzyme consisted of two identically sized subunits, and it contained one pyridoxal phosphate per subunit. The enzyme catalyzed the biosynthesis of cysteine or S-methylcysteine from sulfide or methanethiol and O-acetylserine, respectively, in addition to the formation of S-sulfocysteine from thiosulfate and O-acetylserine. The enzyme is identical to cysteine synthase B. The intracellular level of this enzyme was regulated by lesser extents of the same factors as those effective for cysteine synthase A. Images PMID:6373737

  14. The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives

    PubMed Central

    Liepman, Aaron H.; Cavalier, David M.

    2012-01-01

    The CELLULOSE SYNTHASE (CESA) superfamily of proteins contains several sub-families of closely related CELLULOSE SYNTHASE-LIKE (CSL) sequences. Among these, the CSLA and CSLC families are closely related to each other and are the most evolutionarily divergent from the CESA family. Significant progress has been made with the functional characterization of CSLA and CSLC genes, which have been shown to encode enzymes with 1,4-β-glycan synthase activities involved in the biosynthesis of mannan and possibly xyloglucan backbones, respectively. This review examines recent work on the CSLA and CSLC families from evolutionary, molecular, and biochemical perspectives. We pose a series of questions, whose answers likely will provide further insight about the specific functions of members of the CSLA and CSLC families and about plant polysaccharide biosynthesis is general. PMID:22654891

  15. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    PubMed Central

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  16. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    PubMed

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

  17. Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function

    PubMed Central

    Newman, Adam G.; Vagstad, Anna L.; Belecki, Katherine; Scheerer, Jonathan R.

    2012-01-01

    The polyketide synthase CTB1 is demonstrated to catalyze pyrone formation thereby expanding the known biosynthetic repertoire of thioesterase domains in iterative, non-reducing polyketide synthases. PMID:23108075

  18. The Remarkable Character of Porphobilinogen Synthase.

    PubMed

    Jaffe, Eileen K

    2016-11-15

    Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn(2+); a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb(2+). PBGSs that do not require the soft metal ion Zn(2+) at the active site instead are suspected of using the hard metal Mg(2+). The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg(2+) as an allosteric activator. Mg(2+) functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do

  19. WOW: light print, light propel, light point

    NASA Astrophysics Data System (ADS)

    Glückstad, Jesper; Bañas, Andrew; Aabo, Thomas; Palima, Darwin

    2012-10-01

    We are presenting so-called Wave-guided Optical Waveguides (WOWs) fabricated by two-photon polymerization and capable of being optically manipulated into any arbitrary orientation. By integrating optical waveguides into the structures we have created freestanding waveguides which can be positioned anywhere in a sample at any orientation using real-time 3D optical micromanipulation with six degrees of freedom. One of the key aspects of our demonstrated WOWs is the change in direction of in-coupled light and the marked increase in numerical aperture of the out-coupled light. Hence, each light propelled WOW can tap from a relatively broad incident beam and generate a much more tightly confined light at its tip. The presentation contains both numerical simulations related to the propagation of light through a WOW and preliminary experimental demonstrations on our BioPhotonics Workstation. In a broader context, this research shows that optically trapped micro-fabricated structures can potentially help bridge the diffraction barrier. This structure-mediated paradigm may be carried forward to open new possibilities for exploiting beams from far-field optics down to the sub-wavelength domain.

  20. Nitric oxide synthase (NOS) in the human umbilical cord vessels. An immunohistochemical study.

    PubMed

    Dikranian, K; Trosheva, M; Nikolov, S; Bodin, P

    1994-06-01

    Localization of nitric oxide synthase (NOS) in endothelial cells of umbilical cord vessels and in cultured human umbilical vein endothelial cells was investigated by light and electron-microscopical (immunogold) immunohistochemistry. We observed localization of NOS-immunoreactivity in the majority (97%) of the endothelial cells of the umbilical vein and in a subpopulation (6.7%) of endothelial cells of the umbilical arteries. NOS was observed as well in the amniotic epithelium and in the cells of Wharton's jelly. Immunogold labelling in human umbilical vein endothelial cells dominated in the cellular matrix and was not associated with cellular organelles. Since human umbilical vessels are unique in lacking innervation, the functional significance of endothelium derived relaxing factor EDRF/NO in the local control of vascular flow is discussed.

  1. Emergency Lighting

    NASA Technical Reports Server (NTRS)

    1977-01-01

    A lighting system originally developed for NASA's Apollo and Skylab manned spacecraft resulted in a industrial spinoff and creation of a whole new company to produce and market the product line. The company is UDEC Corp., Waltham, Mass. UDEC's "Multi-Mode" electronic lighting systems are designed for plant emergency and supplemental use, such as night lighting, "always-on" stairwell lights and illuminated exit signs. Their advantages stem from the qualities demanded for spacecraft installation: extremely high fight output with very low energy drain, compactness, light weight, and high reliability. The Multi-Mode system includes long-life fluorescent lamps operated by electronic circuitry, a sealed battery that needs no maintenance for 10 years, and a solid-state battery charger. A typical emergency installation consists of a master module with battery and an eight watt lamp, together with four remote "Satellight" modules powered by the master's battery. As a night lighting system for maintenance or I security, UDEC fixtures can bypass the battery and 1 operate on normal current at a fraction of the energy 1 demand of conventional night lighting. Industrial customers have realized savings of better than ninety percent with UDEC night lights. UDEC started as a basement industry in 1972 but the company has already sold more than 1,000 lighting systems to building operators.

  2. Characterization of spermidine synthase and spermine synthase--The polyamine-synthetic enzymes that induce early flowering in Gentiana triflora.

    PubMed

    Imamura, Tomohiro; Fujita, Kohei; Tasaki, Keisuke; Higuchi, Atsumi; Takahashi, Hideyuki

    2015-08-07

    Polyamines are essential for several living processes in plants. However, regulatory mechanisms of polyamines in herbaceous perennial are almost unknown. Here, we identified homologs of two Arabidopsis polyamine-synthetic enzymes, spermidine synthase (SPDS) and spermine synthase (SPMS) denoted as GtSPDS and GtSPMS, from the gentian plant, Gentiana triflora. Our results showed that recombinant proteins of GtSPDS and GtSPMS possessed SPDS and SPMS activities, respectively. The expression levels of GtSPDS and GtSPMS increased transiently during vegetative to reproductive growth phase and overexpression of the genes hastened flowering, suggesting that these genes are involved in flowering induction in gentian plants.

  3. Characterization of a pine multigene family containing elicitor-responsive stilbene synthase genes.

    PubMed

    Preisig-Müller, R; Schwekendiek, A; Brehm, I; Reif, H J; Kindl, H

    1999-01-01

    Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial beta-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.

  4. Apoplasmic loading in the rice phloem supported by the presence of sucrose synthase and plasma membrane-localized proton pyrophosphatase

    PubMed Central

    Regmi, Kamesh C.; Zhang, Shangji; Gaxiola, Roberto A.

    2016-01-01

    Background and Aims Although Oryza sativa (rice) is one of the most important cereal crops, the mechanism by which sucrose, the major photosynthate, is loaded into its phloem is still a matter of debate. Current opinion holds that the phloem loading pathway in rice could involve either a symplasmic or an apoplasmic route. It was hypothesized, on the basis of a complementary body of evidence from arabidopsis, which is an apoplasmic loader, that the membrane specificity of proton pyrophosphatases (H+-PPases; OVPs) in the sieve element–companion cell (SE-CC) complexes of rice source leaves would support the existence of either of the aforementioned phloem loading mechanisms. Additionally, it was contended that the presence of sucrose synthase in the SE-CC complexes would be consistent with an apoplasmic sucrose loading route in rice. Methods Conventional chemical fixation methods were used for immunohistochemical localization of H+-PPases and sucrose synthase in rice and arabidopsis at the light microscopy level, while ultrastructural immunogold labelling of H+-PPases and sucrose synthase was performed on high-pressure frozen source leaves of rice. Key Results Using immunogold labelling, it was found that OVPs predominantly localize at the plasma membrane (PM) of the SE-CC complexes in rice source leaf minor veins, while in the root meristematic cells, OVPs preferentially localize at the vacuoles. The PM specificity of OPVs in the SE-CC complexes was deemed to support apoplasmic loading in the rice phloem. Further backing for this interpretation came from the sucrose synthase-specific immunogold labelling at the SE-CC complexes of rice source leaves. Conclusion These findings are consistent with the idea that, in the same way as in arabidopsis and a majority of grasses, sucrose is actively loaded into the SE-CC complexes of rice leaves using an apoplasmic step. PMID:26614751

  5. Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases.

    PubMed

    Liu, Benye; Falkenstein-Paul, Hildegard; Schmidt, Werner; Beerhues, Ludger

    2003-06-01

    Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.

  6. The polymorphisms in methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and the risk of colorectal cancer.

    PubMed

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results.

  7. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    PubMed Central

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  8. Sucrose synthase in wild tomato Lycopersicon chmielewskii and tomato fruit sink strength

    Treesearch

    Shi-Jean S. Sung; T. Loboda; S.S. Sung; C.C. Black

    1992-01-01

    Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers.In mature, nongrowing fruits, sucrose synthase activities approached nil values.Therefore, sucrose synthase...

  9. Isoelectric focusing of wound-induced tomato ACC synthase

    SciTech Connect

    White, J.A.; Kende, H. )

    1990-05-01

    Several techniques of electrofocusing have been used to determine whether 1-aminocyclopropane-1-carboxylate (ACC) synthase isolated from wounded tomato pericarp tissue exists in different isoforms, each with its characteristic isoelectric point (pI). The pI of the native enzyme was found to be 6.0 {plus minus} 0.2. When radiolabeled, denatured ACC synthase was electrofocused by non-equilibrium pH gradient electrophoresis (NEpHGE), the enzyme separated into four discernible spots which, upon reaching equilibrium, ranged in pI from 6.6 to 6.9. Immunopurified ACC synthase from four tomato cultivars (Duke, Cornell, Mountain Pride and Pik Red) migrated in each case as a 50-kDa protein on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). We propose that native ACC synthase in extracts of tomato pericarp tissue exists in one single form and that the charge heterogeneities observed upon electrofocusing of denatured enzyme result from modifications of preexisting protein.

  10. Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

    PubMed

    Hagmann, W K; Caldwell, C G; Chen, P; Durette, P L; Esser, C K; Lanza, T J; Kopka, I E; Guthikonda, R; Shah, S K; MacCoss, M; Chabin, R M; Fletcher, D; Grant, S K; Green, B G; Humes, J L; Kelly, T M; Luell, S; Meurer, R; Moore, V; Pacholok, S G; Pavia, T; Williams, H R; Wong, K K

    2000-09-04

    A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

  11. Genetics Home Reference: N-acetylglutamate synthase deficiency

    MedlinePlus

    ... of reactions that occurs in liver cells. This cycle processes excess nitrogen, generated when protein is used by the body, to make a compound called urea that is excreted by the kidneys. The ... cycle. In people with N-acetylglutamate synthase deficiency , N- ...

  12. Biosynthesis of polyketides by trans-AT polyketide synthases.

    PubMed

    Helfrich, Eric J N; Piel, Jörn

    2016-02-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that represents one of the major group of proteins involved in the production of bioactive polyketides. The article includes 609 references and covers the literature from 2009 through June 2015.

  13. Biosynthesis of polyketides by trans-AT polyketide synthases.

    PubMed

    Piel, Jörn

    2010-07-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.

  14. Functional characterization of sesquiterpene synthase from Polygonum minus.

    PubMed

    Ee, Su-Fang; Mohamed-Hussein, Zeti-Azura; Othman, Roohaida; Shaharuddin, Noor Azmi; Ismail, Ismanizan; Zainal, Zamri

    2014-01-01

    Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β -sesquiphellandrene.

  15. Characteristic alatoid 'cineole cassette' monoterpene synthase present in Nicotiana noctiflora.

    PubMed

    Fähnrich, Anke; Neumann, Madeleine; Piechulla, Birgit

    2014-05-01

    Nicotiana species of the section Alatae emit a characteristic floral scent comprising the' cineole cassette' monoterpenes 1,8-cineole, limonene, myrcene, β-pinene, α-pinene, sabinene and α-terpineol. All previously isolated 'cineole cassette'-monoterpene synthase genes are multi product enzymes that synthesize the seven compounds of the 'cineole cassette'. Interestingly, so far this 'alatoid' trait was only shared with the eponymous species Nicotiana suaveolens of the sister section Suaveolentes. To determine the origin of the 'cineole cassette' monoterpene phenotype other potential parent species of section Noctiflorae or Petunoides as well as of the distantly related section Trigonophyllae were analysed. A monoterpene synthase producing the set of 'cineole cassette' compounds was isolated from N. noctiflorae. N. obtusifolia emitted solely 1,8-cineole and no monoterpenes were found in floral scents of N. petunoides and N. palmeri. Interestingly, the phylogenetic analysis clustered the new gene of N. noctiflora closely to the terpineol synthase genes of e.g. N. alata rather than to cineole synthase genes of e.g. N. forgetiana.

  16. Mammalian fatty acid synthase: closure on a textbook mechanism?

    PubMed

    Leadlay, Peter; Baerga-Ortiz, Abel

    2003-02-01

    Mammalian fatty acid synthase is a classic example of a chain-building multienzyme. A cornerstone of its mechanism has been the obligatory collaboration of two identical subunits, with fatty acyl intermediates transferring between them. Now, fresh evidence has upset this view.

  17. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    PubMed Central

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data. PMID:27721652

  18. A Proteolytic Regulator Controlling Chalcone Synthase Stability and Flavonoid Biosynthesis in Arabidopsis

    DOE PAGES

    Zhang, Xuebin; Abrahan, Carolina; Colquhoun, Thomas A.; ...

    2017-04-26

    Flavonoids represent a large family of specialized metabolites involved in plant growth, development, and adaptation. Chalcone synthase (CHS) catalyzes the first step of flavonoid biosynthesis by directing carbon flux from general phenylpropanoid metabolism to flavonoid pathway. Despite extensive characterization of its function and transcriptional regulation, the molecular basis governing its posttranslational modification is enigmatic. Here, we report the discovery of a proteolytic regulator of CHS, namely, KFBCHS, a Kelch domain-containing F-box protein in Arabidopsis thaliana. KFBCHS physically interacts with CHS and specifically mediates its ubiquitination and degradation. KFBCHS exhibits developmental expression patterns in Arabidopsis leaves, stems, and siliques and stronglymore » responds to the dark-to-light (or the light-to-dark) switch, the blue, red, and far-red light signals, and UV-B irradiation. Alteration of KFBCHS expression negatively correlates to the cellular concentration of CHS and the production of flavonoids. Our study suggests that KFBCHS serves as a crucial negative regulator, via mediating CHS degradation, coordinately controlling flavonoid biosynthesis in response to the developmental cues and environmental stimuli.« less

  19. Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

    PubMed Central

    Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

    2009-01-01

    8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

  20. Phytochelatin synthase: of a protease a peptide polymerase made.

    PubMed

    Rea, Philip A

    2012-05-01

    Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes. Copyright © Physiologia Plantarum 2012.

  1. EDITORIAL: Slow light Slow light

    NASA Astrophysics Data System (ADS)

    Boyd, Robert; Hess, Ortwin; Denz, Cornelia; Paspalakis, Emmanuel

    2010-10-01

    Research into slow light began theoretically in 1880 with the paper [1] of H A Lorentz, who is best known for his work on relativity and the speed of light. Experimental work started some 60 years later with the work of S L McCall and E L Hahn [2] who explored non-linear self-induced transparency in ruby. This field of research has burgeoned in the last 10 years, starting with the work of L Vestergaard Hau and coworkers on slow light via electromagnetically induced transparency in a Bose-Einstein condensate [3]. Many groups are now able to slow light down to a few metres per second or even stop the motion of light entirely [4]. Today, slow light - or more often `slow and fast light' - has become its own vibrant field with a strongly increasing number of publications. In broad scope, slow light research can be categorized in terms of the sort of physical mechanism used to slow down the light. One sort of slow light makes use of material dispersion. This dispersion can be the natural dispersion of the ordinary refractive index or can be the frequency dependence of some nonlinear optical process, such as electromagnetically induced transparency, coherent population oscillations, stimulated light scattering, or four-wave mixing processes. The second sort of slow light makes use of the wavelength dependence of artificially structured materials, such as photonic crystals, optical waveguides, and collections of microresonators. Material systems in which slow light has been observed include metal vapours, rare-earth-doped materials, Raman and Brillioun gain media, photonic crystals, microresonators and, more recently, metamaterials. A common feature of all of these schemes is the presence of a sharp single resonance or multiple resonances produced by an atomic transition, a resonance in a photonic structure, or in a nonlinear optical process. Current applications of slow light include a series of attractive topics in optical information processing, such as optical data

  2. Transgene silencing of sucrose synthase in alfalfa stem vascular tissue by a truncated phosphoenolpyruvate carboxylase: sucrose synthase construct

    USDA-ARS?s Scientific Manuscript database

    An important role of sucrose synthase (SUS, EC 2.4.1.13) in plants is to provide UDP-glucose needed for cellulose synthesis in cell walls. We examined if over-expressing SUS in alfalfa (Medicago sativa L.) would increase cellulose content of stem cell walls. Alfalfa plants were transformed with two ...

  3. Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

    PubMed

    Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

    2014-01-01

    In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

  4. Mechanism-oriented redesign of an isomaltulose synthase to an isomelezitose synthase by site-directed mutagenesis.

    PubMed

    Görl, Julian; Timm, Malte; Seibel, Jürgen

    2012-01-02

    An isomelezitose synthase was redesigned out of the sucrose isomerase from Protaminobacter rubrum for the synthesis of isomelezitose (6-O(F)-glucosylsucrose), a potential nutraceutical. The variants F297A, F297P, R333K, F321A_F319A and E428D catalyze the formation of isomelezitose in up to 70 % yield.

  5. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    SciTech Connect

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  6. ATP synthase repression in tobacco restricts photosynthetic electron transport, CO2 assimilation, and plant growth by overacidification of the thylakoid lumen.

    PubMed

    Rott, Markus; Martins, Nádia F; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Kramer, David M; Schöttler, Mark A

    2011-01-01

    Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b(6)f complex to the metabolic demand for ATP and NADPH. While the cytochrome b(6)f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b(6)f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO(2) fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed.

  7. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa.

    PubMed

    Taura, Futoshi; Sirikantaramas, Supaart; Shoyama, Yoshinari; Yoshikai, Kazuyoshi; Shoyama, Yukihiro; Morimoto, Satoshi

    2007-06-26

    Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants.

  8. Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens.

    PubMed

    Roeder, Susanna; Hartmann, Anna-Maria; Effmert, Uta; Piechulla, Birgit

    2007-09-01

    The white flowers of N. suaveolens emit a complex bouquet of fragrance volatiles. The dominant compounds are benzenoids (e.g. methyl benzoate, methyl salicylate, benzyl benzoate and benzyl salicylate), monoterpenes (1,8-cineole, limonene, sabinene, E-beta-ocimene, beta-beta-myrcene, alpha- and beta-pinene and alpha-terpineole) and sesquiterpenes (e.g. caryophyllene), which are all emitted at higher levels during the night. Here, we show that the simultaneous nocturnal emission of most monoterpenes is realized by a single floral-specific multi-product enzyme (1,8-cineole synthase, CIN), which synthesizes the monoterpenes of the "cineole cassette". Interestingly, N. suaveolens is the only known taxon of the Suaveolentes section to have a flower emitting "cineole cassette of monoterpenes" which is otherwise typical for the Alatae section. Gene sequence analysis of CIN has revealed the highest similarities to other angiosperm monoterpene synthases from Vitis vinifera, Quercus ilex, Citrus unshiu and C. limon, which cluster in the same branch of the terpene synthase B subfamily. However, based on its synthesized products, N. suaveolens CIN shares similarity with enzymes of the Arabidopsis thaliana root and Salvia officinalis leaf. The N. suaveolens CIN gene is only expressed in the stigma/style tissue and petals. Thin sections of petals present the enzyme primarily in the adaxial and abaxial epidermis; this facilitates the comprehensive emission of volatiles in all spacial directions. The oscillation of monoterpene emission is a consequence of the regulation of the CIN gene by the circadian clock, with oscillations occurring at the level of transcript and protein accumulations and of enzyme activity. Light/dark or dark/light transition signals synchronize the slow-running endogenous clock. Two strategies for synchronized scent emission have been established in N. suaveolens flowers: (i) the synthesis of volatile organic compounds by a multi-product enzyme and (ii) the

  9. Light Learning.

    ERIC Educational Resources Information Center

    Cutshall, Sandy

    2002-01-01

    Describes a career and technical education program on photonics, the study, research, and development of equipment and concepts used in the transmission of information through light, including fiber optics and experimental laser technologies. (JOW)

  10. Combination Light

    NASA Astrophysics Data System (ADS)

    1990-01-01

    The Rayovac TANDEM is an advanced technology combination work light and general purpose flashlight that incorporates several NASA technologies. The TANDEM functions as two lights in one. It features a long range spotlight and wide angle floodlight; simple one-hand electrical switching changes the beam from spot to flood. TANDEM developers made particular use of NASA's extensive research in ergonomics in the TANDEM's angled handle, convenient shape and different orientations. The shatterproof, water resistant plastic casing also draws on NASA technology, as does the shape and beam distance of the square diffused flood. TANDEM's heavy duty magnet that permits the light to be affixed to any metal object borrows from NASA research on rare earth magnets that combine strong magnetic capability with low cost. Developers used a NASA-developed ultrasonic welding technique in the light's interior.

  11. Light Learning.

    ERIC Educational Resources Information Center

    Cutshall, Sandy

    2002-01-01

    Describes a career and technical education program on photonics, the study, research, and development of equipment and concepts used in the transmission of information through light, including fiber optics and experimental laser technologies. (JOW)

  12. Circadian light

    PubMed Central

    2010-01-01

    The present paper reflects a work in progress toward a definition of circadian light, one that should be informed by the thoughtful, century-old evolution of our present definition of light as a stimulus for the human visual system. This work in progress is based upon the functional relationship between optical radiation and its effects on nocturnal melatonin suppression, in large part because the basic data are available in the literature. Discussed here are the fundamental differences between responses by the visual and circadian systems to optical radiation. Brief reviews of photometry, colorimetry, and brightness perception are presented as a foundation for the discussion of circadian light. Finally, circadian light (CLA) and circadian stimulus (CS) calculation procedures based on a published mathematical model of human circadian phototransduction are presented with an example. PMID:20377841

  13. Light's twist

    PubMed Central

    Padgett, Miles

    2014-01-01

    That light travels in straight lines is a statement of the obvious. However, the energy and momentum flow within light beams can twist to form vortices such as eddies in a stream. These twists carry angular momentum, which can make microscopic objects spin, be used to encode extra information in communication systems, enable the design of novel imaging systems and allow new tests of quantum mechanics. PMID:25484612

  14. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    PubMed Central

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  15. Kinetic mechanism of rabbit muscle glycogen synthase I.

    PubMed

    Gold, A M

    1980-08-05

    The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [Sølling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.

  16. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

    PubMed

    Grundy, Daniel J; Chen, Mengbin; González, Verónica; Leoni, Stefano; Miller, David J; Christianson, David W; Allemann, Rudolf K

    2016-04-12

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues

  17. Hematopoetic Prostaglandin D Synthase: An ESR1-Dependent Oviductal Epithelial Cell Synthase

    PubMed Central

    Jeoung, Myoungkun; Shim, Sarah; Park, Ji Yeon; Lee, Jae Eun; Sapsford, Lindsay A.; Trudgen, Kourtney; Ko, Chemyong; Gye, Myung Chan; Jo, Misung

    2012-01-01

    Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD2: PGD2 receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct. PMID:22374975

  18. Deficiency of sphingomyelin synthase-1 but not sphingomyelin synthase-2 causes hearing impairments in mice

    PubMed Central

    Lu, Mei-Hong; Takemoto, Makoto; Watanabe, Ken; Luo, Huan; Nishimura, Masataka; Yano, Masato; Tomimoto, Hidekazu; Okazaki, Toshiro; Oike, Yuichi; Song, Wen-Jie

    2012-01-01

    Sphingomyelin (SM) is a sphingolipid reported to function as a structural component of plasma membranes and to participate in signal transduction. The role of SM metabolism in the process of hearing remains controversial. Here, we examined the role of SM synthase (SMS), which is subcategorized into the family members SMS1 and SMS2, in auditory function. Measurements of auditory brainstem response (ABR) revealed hearing impairment in SMS1−/− mice in a low frequency range (4–16 kHz). As a possible mechanism of this impairment, we found that the stria vascularis (SV) in these mice exhibited atrophy and disorganized marginal cells. Consequently, SMS1−/− mice exhibited significantly smaller endocochlear potentials (EPs). As a possible mechanism for EP reduction, we found altered expression patterns and a reduced level of KCNQ1 channel protein in the SV of SMS1−/− mice. These mice also exhibited reduced levels of distortion product otoacoustic emissions. Quantitative comparison of the SV atrophy, KCNQ1 expression, and outer hair cell density at the cochlear apical and basal turns revealed no location dependence, but more macrophage invasion into the SV was observed in the apical region than the basal region, suggesting a role of cochlear location-dependent oxidative stress in producing the frequency dependence of hearing loss in SMS1−/− mice. Elevated ABR thresholds, decreased EPs, and abnormal KCNQ1 expression patterns in SMS1−/− mice were all found to be progressive with age. Mice lacking SMS2, however, exhibited neither detectable hearing loss nor changes in their EPs. Taken together, our results suggest that hearing impairments occur in SMS1−/− but not SMS2−/− mice. Defects in the SV with subsequent reductions in EPs together with hair cell dysfunction may account, at least partially, for hearing impairments in SMS1−/− mice. PMID:22641779

  19. Deficiency of sphingomyelin synthase-1 but not sphingomyelin synthase-2 causes hearing impairments in mice.

    PubMed

    Lu, Mei-Hong; Takemoto, Makoto; Watanabe, Ken; Luo, Huan; Nishimura, Masataka; Yano, Masato; Tomimoto, Hidekazu; Okazaki, Toshiro; Oike, Yuichi; Song, Wen-Jie

    2012-08-15

    Sphingomyelin (SM) is a sphingolipid reported to function as a structural component of plasma membranes and to participate in signal transduction. The role of SM metabolism in the process of hearing remains controversial. Here, we examined the role of SM synthase (SMS), which is subcategorized into the family members SMS1 and SMS2, in auditory function. Measurements of auditory brainstem response (ABR) revealed hearing impairment in SMS1−/− mice in a low frequency range (4–16 kHz). As a possible mechanism of this impairment, we found that the stria vascularis (SV) in these mice exhibited atrophy and disorganized marginal cells. Consequently, SMS1−/− mice exhibited significantly smaller endocochlear potentials (EPs). As a possible mechanism for EP reduction, we found altered expression patterns and a reduced level of KCNQ1 channel protein in the SV of SMS1−/− mice. These mice also exhibited reduced levels of distortion product otoacoustic emissions. Quantitative comparison of the SV atrophy, KCNQ1 expression, and outer hair cell density at the cochlear apical and basal turns revealed no location dependence, but more macrophage invasion into the SV was observed in the apical region than the basal region, suggesting a role of cochlear location-dependent oxidative stress in producing the frequency dependence of hearing loss in SMS1−/− mice. Elevated ABR thresholds, decreased EPs, and abnormal KCNQ1 expression patterns in SMS1−/− mice were all found to be progressive with age. Mice lacking SMS2, however, exhibited neither detectable hearing loss nor changes in their EPs. Taken together, our results suggest that hearing impairments occur in SMS1−/− but not SMS2−/− mice. Defects in the SV with subsequent reductions in EPs together with hair cell dysfunction may account, at least partially, for hearing impairments in SMS1−/− mice.

  20. Ectopic expression of ceramide synthase 2 in neurons suppresses neurodegeneration induced by ceramide synthase 1 deficiency

    PubMed Central

    Spassieva, Stefka D.; Ji, Xiaojie; Liu, Ye; Gable, Kenneth; Bielawski, Jacek; Dunn, Teresa M.; Bieberich, Erhard; Zhao, Lihong

    2016-01-01

    Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22–C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypothesis, we found that only LCBs, the substrates common for all of the CerS isoforms, but not ceramides and complex sphingolipids, were restored to the wild-type levels in the Cers2-rescued Cers1 mutant mouse brains. Furthermore, LCBs induced neurite fragmentation in cultured neurons at concentrations corresponding to the elevated levels in the CerS1-deficient brain. The strong association of LCB levels with neuronal survival both in vivo and in vitro suggests high-level accumulation of LCBs is a possible underlying cause of the CerS1 deficiency-induced neuronal death. PMID:27162368

  1. A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase.

    PubMed

    Win, N H; Ishikawa, T; Saito, N; Kato, M; Yokokura, H; Watanabe, Y; Iida, Y; Hidaka, H

    1996-03-28

    HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide

  2. Carbon Monoxide Dehydrogenases and Acetyl-CoA Synthases: Light at the End of the Tunnel?

    SciTech Connect

    Paul A. Lindahl

    2002-02-19

    OAK-B135 Metalloenzymes seem to ''come of age'' when their structures are known at atomic resolution, spectroscopic and catalytic properties are basically understood, and genetic expression systems are available. Such foundations allow detailed mechanistic and spectroscopic properties to be probed and correlated to structure. The objective of this article is to summarize what is known about the title group of enzymes, and show that, to a large degree, they have come of age.

  3. Defining the Product Chemical Space of Monoterpenoid Synthases

    PubMed Central

    Tian, Boxue; Poulter, C. Dale; Jacobson, Matthew P.

    2016-01-01

    Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes. PMID:27517297

  4. Sucrose Synthase Expression during Cold Acclimation in Wheat 1

    PubMed Central

    Crespi, Martin D.; Zabaleta, Eduardo J.; Pontis, Horacio G.; Salerno, Graciela L.

    1991-01-01

    When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)+ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress. ImagesFigure 1Figure 2Figure 3 PMID:16668270

  5. Structure of isochorismate synthase DhbC from Bacillus anthracis.

    PubMed

    Domagalski, M J; Tkaczuk, K L; Chruszcz, M; Skarina, T; Onopriyenko, O; Cymborowski, M; Grabowski, M; Savchenko, A; Minor, W

    2013-09-01

    The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg(2+)-dependent catalytic mechanism.

  6. Structure of isochorismate synthase DhbC from Bacillus anthracis

    PubMed Central

    Domagalski, M. J.; Tkaczuk, K. L.; Chruszcz, M.; Skarina, T.; Onopriyenko, O.; Cymborowski, M.; Grabowski, M.; Savchenko, A.; Minor, W.

    2013-01-01

    The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg2+-dependent catalytic mechanism. PMID:23989140

  7. Synchrotron light

    SciTech Connect

    Craievich, A. )

    1990-01-01

    Several developed countries such as the USA, URSS, England, France, Italy, Sweden and Japan have one or more of these synchrotron light facilities operating or under construction. Some developing countries have constructed (China) or are building (Taiwan, India, Korea, Brazil) synchrotron light facilities. The construction of the Brazilian synchrotron source began in June, 1987. After two years of activities, the injector linac for the electron storage ring is in its final stage of construction. These Proceedings contain the Invited Lectures presented at the Workshop by specialists working on synchrotron light applications and related instrumentation and by members of LNLS regarding technical details of the Brazilian project. The II Workshop Synchrotron Light: Applications and Related Instrumentation was dedicated to oral presentations about applications of synchrotron light, most of which were not covered during the I Workshop, organized by LNLS in 1988, and the Proceedings of which were published by World Scientific. The II Workshop included discussions on the application possibilities for the newly designed LNLS 1.15 GeV storage ring, and on the modifications which would eventually be necessary for the work-station and instrumentation projects currently in progress at LNLS and at various external user laboratories.

  8. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    PubMed

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

  9. Phylogenetic relationships of rhizobia based on citrate synthase gene sequences.

    PubMed

    Hernández-Lucas, Ismael; Rogel-Hernández, Marco Antonio; Segovia, Lorenzo; Rojas-Jiménez, Keilor; Martínez-Romero, Esperanza

    2004-11-01

    Partial nucleotide sequences of the citrate synthase (gltA) gene from different rhizobia genera were determined. Tree topologies based on this housekeeping gene were similar to that obtained using 16S rRNA sequences. However gltA appeared to be more reliable at determining phylogenetic relationships of closely related taxa. We propose gltA sequences as an additional tool to be used in molecular phylogenetic studies.

  10. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, Eran

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  11. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, E.

    1998-12-15

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

  12. Piriformospora indica requires kaurene synthase activity for successful plant colonization.

    PubMed

    Li, Liang; Chen, Xi; Ma, Chaoyang; Wu, Hongqing; Qi, Shuting

    2016-05-01

    Ent-kaurene (KS) synthases and ent-kaurene-like (KSL) synthases are involved in the biosynthesis of phytoalexins and/or gibberellins which play a role in plant immunity and development. The relationship between expression of five synthase genes (HvKSL1, HvKS2, HvKS4, HvKS5, HvKSL4) and plant colonization by the endophytic fungus Piriformospora indica was assessed in barley (Hordeum vulgare). The KS gene family is differently up-regulated at 1, 3 and 7 day after P. indica inoculation. By comparison, the HvKSL4 gene expression pattern is more significantly affected by UV irradiation and P. indica colonization. The characterizations of two silencing lines (HvKSL1-RNAi, HvKSL4-RNAi) also were analyzed. HvKSL1-RNAi and HvKSL4-RNAi lines in the first generation lead to less dark green leaves and slower plant development. Further, reduced spikelet fertility in progenies of RNAi plants heterozygous for HvKSL1 were observed, but not for HvKSL4. T2 generation of HvKSL1-RNAi line showed semi-dwarf phenotype while the wild type phenotype could be restored by applying GA3. Silencing of HvKSL4 and HvKSL1 resulted in reduced colonization by P. indica especially in the HvKSL1-RNAi line. These results probably suggest the presence of two ent-KS synthase in barley, one (HvKSL1) that participates in the biosynthesis of GAs and another (HvKSL4) that is involved in the biosynthesis of phytoalexins.

  13. Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases1

    PubMed Central

    Pandith, Shahzad A.; Dhar, Niha; Bhat, Wajid Waheed; Kushwaha, Manoj; Gupta, Ajai P.; Shah, Manzoor A.; Vishwakarma, Ram

    2016-01-01

    Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and

  14. Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders.

    PubMed

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

    Prostaglandin (PG)E2 encompasses crucial roles in pain, fever, inflammation and diseases with inflammatory component, such as cancer, but is also essential for gastric, renal, cardiovascular and immune homeostasis. Cyclooxygenases (COX) convert arachidonic acid to the intermediate PGH2 which is isomerized to PGE2 by at least three different PGE2 synthases. Inhibitors of COX - non-steroidal anti-inflammatory drugs (NSAIDs) - are currently the only available therapeutics that target PGE2 biosynthesis. Due to adverse effects of COX inhibitors on the cardiovascular system (COX-2-selective), stomach and kidney (COX-1/2-unselective), novel pharmacological strategies are in demand. The inducible microsomal PGE2 synthase (mPGES)-1 is considered mainly responsible for the excessive PGE2 synthesis during inflammation and was suggested as promising drug target for suppressing PGE2 biosynthesis. However, 15 years after intensive research on the biology and pharmacology of mPGES-1, the therapeutic value of mPGES-1 as drug target is still vague and mPGES-1 inhibitors did not enter the market so far. This commentary will first shed light on the structure, mechanism and regulation of mPGES-1 and will then discuss its biological function and the consequence of its inhibition for the dynamic network of eicosanoids. Moreover, we (i) present current strategies for interfering with mPGES-1-mediated PGE2 synthesis, (ii) summarize bioanalytical approaches for mPGES-1 drug discovery and (iii) describe preclinical test systems for the characterization of mPGES-1 inhibitors. The pharmacological potential of selective mPGES-1 inhibitor classes as well as dual mPGES-1/5-lipoxygenase inhibitors is reviewed and pitfalls in their development, including species discrepancies and loss of in vivo activity, are discussed.

  15. The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

    PubMed Central

    1991-01-01

    The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells. PMID:2050738

  16. Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice

    PubMed Central

    Li, Zhen; Wang, Qingguo; Yao, Fangyin; Yang, Lianqun; Pan, Jiaowen; Liu, Wei

    2015-01-01

    Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 μg/g FW in seedlings and 3.053 μg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C) or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering. PMID:26302213

  17. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    PubMed

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  18. From bacterial to human dihydrouridine synthase: automated structure determination

    SciTech Connect

    Whelan, Fiona Jenkins, Huw T.; Griffiths, Samuel C.; Byrne, Robert T.; Dodson, Eleanor J.; Antson, Alfred A.

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  19. Effect of calcofluor white on chitin synthases from Saccharomyces cerevisiae.

    PubMed Central

    Roncero, C; Valdivieso, M H; Ribas, J C; Durán, A

    1988-01-01

    The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction of Calcofluor with growing cells had to occur at around pH 6. Treatment of growing cells from these strains with the fluorochrome led to an increase in the total levels of Chs1 and Chs2 activities measured on permeabilized cells. During treatment, basal levels (activities expressed in the absence of exogenous proteolytic activation) of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide. During alpha-factor treatment, both Chs1 and Chs2 levels increased; however, as opposed to what occurred during the mitotic cell cycle, there was no further increase in Chs1 or Chs2 activities by Calcofluor treatment. Images PMID:2965145

  20. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    SciTech Connect

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  1. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    PubMed

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

  2. Iterative Polyketide Biosynthesis by Modular Polyketide Synthases in Bacteria

    PubMed Central

    Chen, Haotong; Du, Liangcheng

    2015-01-01

    Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases has a characteristic modular organization, and each module within a PKS carries out one cycle of polyketide chain elongation; thus each module is “non-iterative” in function. It was possible to predict the basic structure of a polyketide product from the module organization of the PKSs, since there generally existed a co-linearity between the number of modules and the number of chain elongations. However, more and more bacterial modular PKSs fail to conform to the “canonical rules”, and a particularly noteworthy group of non-canonical PKSs is the bacterial iterative type I PKSs. This review covers recent examples of iteratively-used modular PKSs in bacteria. These non-canonical PKSs give rise to a large array of natural products with impressive structural diversity. The molecular mechanism behind the iterations is often unclear, presenting a new challenge to the rational engineering of these PKSs with the goal of generating new natural products. Structural elucidation of these synthase complexes and better understanding of potential PKS-PKS interactions as well as PKS-substrate recognition may provide new prospects and inspirations for the discovery and engineering of new bioactive polyketides. PMID:26549236

  3. The structural basis of Erwinia rhapontici isomaltulose synthase.

    PubMed

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

  4. Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase.

    PubMed

    Carman, George M; Han, Gil-Soo

    2017-08-16

    The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

    PubMed Central

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations. PMID:24069347

  6. [Progress and application prospects of glutamine synthase in plants].

    PubMed

    Feng, Wanjun; Xing, Guofang; Niu, Xulong; Dou, Chen; Han, Yuanhuai

    2015-09-01

    Nitrogen is one of the most important nutrient elements for plants and a major limiting factor in plant growth and crop productivity. Glutamine synthase (GS) is a key enzyme involved in the nitrogen assimilation and recycling in plants. So far, members of the glutamine synthase gene family have been characterized in many plants such as Arabidopsis, rice, wheat, and maize. Reports show that GS are involved in the growth and development of plants, in particular its role in seed production. However, the outcome has generally been inconsistent, which are probably derived from the transcriptional and post-translational regulation of GS genes. In this review, we outlined studies on GS gene classification, QTL mapping, the relationship between GS genes and plant growth with nitrogen and the distribution characters, the biological functions of GS genes, as well as expression control at different regulation levels. In addition, we summarized the application prospects of glutamine synthetase genes in enhancing plant growth and yield by improving the nitrogen use efficiency. The prospects were presented on the improvement of nitrogen utility efficiency in crops and plant nitrogen status diagnosis on the basis of glutamine synthase gene regulation.

  7. Regulation of expression, activity and localization of fungal chitin synthases

    PubMed Central

    Rogg, Luise E.; Fortwendel, Jarrod R.; Juvvadi, Praveen R.; Steinbach, William J.

    2013-01-01

    The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of β-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets. PMID:21526913

  8. Virus-Induced Silencing of a Plant Cellulose Synthase Gene

    PubMed Central

    Burton, Rachel A.; Gibeaut, David M.; Bacic, Antony; Findlay, Kim; Roberts, Keith; Hamilton, Andrew; Baulcombe, David C.; Fincher, Geoffrey B.

    2000-01-01

    Specific cDNA fragments corresponding to putative cellulose synthase genes (CesA) were inserted into potato virus X vectors for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing. Plants infected with one group of cDNAs had much shorter internode lengths, small leaves, and a “dwarf” phenotype. Consistent with a loss of cell wall cellulose, abnormally large and in many cases spherical cells ballooned from the undersurfaces of leaves, particularly in regions adjacent to vascular tissues. Linkage analyses of wall polysaccharides prepared from infected leaves revealed a 25% decrease in cellulose content. Transcript levels for at least one member of the CesA cellulose synthase gene family were lower in infected plants. The decrease in cellulose content in cell walls was offset by an increase in homogalacturonan, in which the degree of esterification of carboxyl groups decreased from ∼50 to ∼33%. The results suggest that feedback loops interconnect the cellular machinery controlling cellulose and pectin biosynthesis. On the basis of the phenotypic features of the infected plants, changes in wall composition, and the reduced abundance of CesA mRNA, we concluded that the cDNA fragments silenced one or more cellulose synthase genes. PMID:10810144

  9. Rotation and structure of FoF1-ATP synthase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

  10. The Spatial Distribution of Sucrose Synthase Isozymes in Barley.

    PubMed Central

    Guerin, J.; Carbonero, P.

    1997-01-01

    The sucrose (Suc) synthase enzyme purified from barley (Hordeum vulgare L.) roots is a homotetramer that is composed of 90-kD type 1 Suc synthase (SS1) subunits. Km values for Suc and UDP were 30 mM and 5 [mu]M, respectively. This enzyme can also utilize ADP at 25% of the UDP rate. Anti-SS1 polyclonal antibodies, which recognized both SS1 and type 2 Suc synthase (SS2) (88-kD) subunits, and antibodies raised against a synthetic peptide, LANGSTDNNFV, which were specific for SS2, were used to study the spatial distribution of these subunits by immunoblot analysis and immunolocalization. Both SS1 and SS2 were abundantly expressed in endosperm, where they polymerize to form the five possible homo- and heterotetramers. Only SS1 homotetramers were detected in young leaves, where they appeared exclusively in phloem cells, and in roots, where expression was associated with cap cells and the vascular bundle. In the seed both SS1 and SS2 were present in endosperm, but only SS1 was apparent in the chalazal region, the nucellar projection, and the vascular bundle. The physiological implications for the difference in expression patterns observed are discussed with respect to the maize (Zea mays L.) model. PMID:12223688

  11. Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase.

    PubMed

    Zomer, A W; Michels, P A; Opperdoes, F R

    1999-10-25

    Alkyl dihydroxyacetone-phosphate synthase is the second enzyme of the ether-lipid biosynthetic pathway which is responsible for the introduction of the ether linkage between a fatty alcohol and a glycerol present in a subclass of phospholipids, the plasmalogens and possibly in glycolipid membrane anchors. In this study the gene coding for alkyl dihydroxyacetone-phosphate synthase was isolated from Trypanosoma brucei. Southern blot analysis of total genomic DNA suggested the presence of a single copy gene. The analysis, together with sequencing of different cDNA clones showed that the two alleles of the gene differ in only one nucleotide. The gene encodes a protein of 612 amino acids with a calculated molecular mass of 68,891, not counting the initiator methionine. It carries a type-1 peroxisomal targeting signal (a C-terminal tripeptide--AHL) and a calculated overall positive charge of +10. The gene was expressed in a bacterial system and the corresponding protein carrying a His-tag was purified. The recombinant alkyl dihydroxyacetone-phosphate synthase and the enzyme isolated directly from the glycosomes of bloodstream-form trypanosomes have comparable kinetics. The Km for hexadecanol was 42 microM, while approximately 100 microM of palmitoyl dihydroxyacetone phosphate (DHAP) was necessary for optimal activity. Sodium chloride inhibited both the His-tagged protein and the enzyme isolated from the glycosomes of bloodstream-form and insect stage T. brucei.

  12. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    PubMed Central

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  13. Light's Darkness

    ScienceCinema

    Padgett, Miles [University of Glasgow, Glasgow, Scotland

    2016-07-12

    Optical vortices and orbital angular momentum are currently topical subjects in the optics literature. Although seemingly esoteric, they are, in fact, the generic state of light and arise whenever three or more plane waves interfere. To be observed by eye the light must be monochromatic. Laser speckle is one such example, where the optical energy circulates around each black spot, giving a local orbital angular momentum. This talk with report three on-going studies. First, when considering a volume of interfering waves, the laser specs map out threads of complete darkness embedded in the light. Do these threads form loops? Links? Or even knots? Second, when looking through a rapidly spinning window, the image of the world on the other side is rotated: true or false? Finally, the entanglement of orbital angular momentum states means measuring how the angular position of one photons sets the angular momentum of another: is this an angular version of the EPR (Einstein, Podolsky, and Rosen) paradox?

  14. Lighting installations

    NASA Technical Reports Server (NTRS)

    Schurer, Kees

    1994-01-01

    Model computations that give the lay-out of a lighting installation have to be implemented in the real world. There, deviations from the ideal performance of just about every element of the installation will be felt. A list of possible sources of non-ideal behavior, based on practical experience, are: lamps, ballasts, reflectors, mounting position, sagging of lamps, and soiling. It is clear that with all possible deviations from the ideal the homogeneity of a real lighting installation can never be as good as the one computed. The only way to make sure it is nearly as good is by measurement of the actual light distribution. Then, an occasional adjustment or replacement may often yield a satisfactory result. This measurement should really be part of the installation contract.

  15. Screening for latent acute intermittent porphyria: the value of measuring both leucocyte delta-aminolaevulinic acid synthase and erythrocyte uroporphyrinogen-1-synthase activities.

    PubMed Central

    McColl, K E; Moore, M R; Thompson, G G; Goldberg, A

    1982-01-01

    Acute intermittent porphyria (AIP) is an autosomal dominantly inherited disorder of haem biosynthesis characterised by reduced activity of the enzyme uroporphyrinogen-1-(URO) synthase and compensatory increased activity of the rate controlling enzyme delta-aminolaevulinic acid (ALA) synthase. Subjects with the disorder should be identified as they are at risk of developing severe porphyric attacks if exposed to a variety of drugs or chemicals. We have assessed the value of measuring the activities of ALA synthase and URO synthase in peripheral blood cells as a means of identifying latent cases in affected families. In AIP subjects, ALA synthase activity was increased and URO synthase decreased compared to controls, through there was considerable overlap between the two groups when either enzyme was examined alone. When both enzymes were examined together, all but one of the 19 AIP patients had both increased ALA synthase activity (greater than 250 nmol ALA/g protein/h) and reduced URO synthase activity (less than 25.1 nmol URO/l RBC/h), whereas none of the 62 controls showed this enzyme pattern. Examination of 35 asymptomatic first degree blood relatives of AIP patients showed that 17 (49%) had the porphyric enzyme pattern with no sex bias. The combined study of these two enzymes permits accurate detection of latent cases of AIP and confirms its autosomal dominant inheritance. PMID:7120315

  16. Interaction between PVY HC-Pro and the NtCF1β-subunit reduces the amount of chloroplast ATP synthase in virus-infected tobacco

    PubMed Central

    Tu, Yayi; Jin, Yongsheng; Ma, Dongyuan; Li, Heng; Zhang, Zhenqian; Dong, Jiangli; Wang, Tao

    2015-01-01

    The photosynthetic rate of virus-infected plants is always reduced. However, the molecular mechanism underlying this phenomenon remains unclear. The helper component-proteinase (HC-Pro) of Potato virus Y (PVY) was found in the chloroplasts of PVY-infected tobacco, indicating some new function of HC-Pro in the chloroplasts. We generated HC-Pro transgenic plants with a transit peptide to target the protein to chloroplast. The HC-Pro transgenic tobacco showed a decreased photosynthetic rate by 25% at the light intensity of 600 μmol m−2 s−1. Using a yeast two-hybrid screening assay to search for chloroplast proteins interacting with HC-Pro, we identified that PVY HC-Pro can interact with the chloroplast ATP synthase NtCF1β-subunit. This interaction was confirmed by GST pull-down and co-immunoprecipitation assays. HC-Pro didn’t interfere with the activity of assembled ATP synthase in vitro. The HC-Pro/NtCF1β-subunit interaction might affect the assembly of ATP synthase complex. Quantitative western blot and immunogold labeling of the ATP synthase indicated that the amount of ATP synthase complex was decreased in both the HC-Pro transgenic and the PVY-infected tobacco. These results demonstrate that HC-Pro plays an important role in reducing the photosynthetic rate of PVY-infected plants, which is a completely new role of HC-Pro besides its multiple known functions. PMID:26499367

  17. Diel activity of sucrose phosphate synthase in rice. [Oryza sativa L

    SciTech Connect

    Hussain, M.W.; Bowes, G.; Rowland-Bamford, A.J.; Allen, L.H. )

    1991-05-01

    Rice (Oryza sativa L.) was grown in growth chambers at 28/23C day/night temperatures with 16-h photoperiod at 600 umol m{sup {minus}2} s{sup {minus}1}. Diel sucrose phosphate synthase (SPS) activity, at 21 days after planting, was measured at saturating substrate concentrations. Data suggests that SPS activity increased during illumination to a maximum of 0.8 nmol mg{sup {minus}1} protein min{sup {minus}1} after 5h. Throughout the remainder of the light period there was a slow decline in activity. Upon darkening, activity further declined to 0.4 nmol mg{sup {minus}1} protein min{sup {minus}1}, a basal level that was maintained throughout the night. It appears that rice SPS undergoes light/dark transitions, suggesting there may be two kinetic forms of SPS. Changes in SPS activity will be discussed in relation to kinetic studies, and also CO{sub 2} enrichment of rice during growth.

  18. Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation. [Spinacia oleracea

    SciTech Connect

    Huber, J.L. ); Huber, S.C. North Carolina State Univ., Raleigh ); Hite, D.R.C.; Outlaw, W.H. Jr. )

    1991-01-01

    Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ({gamma}-{sup 32}P)ATP. Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25{degrees}C before assay. The spontaneous inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg{sup 2+} and was relatively insensitive to Ca{sup 2+} and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with ({sup 32}P)Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate.

  19. [BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

    PubMed

    Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

    2015-01-01

    A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

  20. Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

    PubMed Central

    Koo, Hyun Jo; Gang, David R.

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

  1. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

    PubMed

    Shibuya, M; Zhang, H; Endo, A; Shishikura, K; Kushiro, T; Ebizuka, Y

    1999-11-01

    Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.

  2. Polymorphous light eruption

    MedlinePlus

    Polymorphous light eruption (PMLE) is a common skin reaction in people who are sensitive to sunlight (ultraviolet light). ... Polymorphic light eruption; Photodermatosis; PMLE; Benign summer light eruption

  3. Chloropropionyl-CoA: a mechanism-based inhibitor of HMG-CoA synthase and fatty acid synthase

    SciTech Connect

    Miziorko, H.M.; Ahmad, F.; Behnke, C.E.

    1986-05-01

    Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-(/sup 3/H)prop-CoA is incubated with the target enzymes, /sup 3/H/sub 2/O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ..-->.. infinity) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k/sub 2/ = 86.7M/sup -1/min/sup -1/ at 30/sup 0/, pH 7.0).

  4. β-Cyanoalanine Synthase Is a Mitochondrial Cysteine Synthase-Like Protein in Spinach and Arabidopsis1

    PubMed Central

    Hatzfeld, Yves; Maruyama, Akiko; Schmidt, Ahlert; Noji, Masaaki; Ishizawa, Kimiharu; Saito, Kazuki

    2000-01-01

    β-Cyano-alanine synthase (CAS; EC 4.4.1.9) plays an important role in cyanide metabolism in plants. Although the enzymatic activity of β-cyano-Ala synthase has been detected in a variety of plants, no cDNA or gene has been identified so far. We hypothesized that the mitochondrial cysteine synthase (CS; EC 4.2.99.8) isoform, Bsas3, could actually be identical to CAS in spinach (Spinacia oleracea) and Arabidopsis. An Arabidopsis expressed sequence tag database was searched for putative Bsas3 homologs and four new CS-like isoforms, ARAth;Bsas1;1, ARAth;Bsas3;1, ARAth;Bsas4;1, and ARAth;Bsas4;2, were identified in the process. ARAth;Bsas3;1 protein was homologous to the mitochondrial SPIol;Bsas3;1 isoform from spinach, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2 proteins defined a new class within the CS-like proteins family. In contrast to spinach SPIol;Bsas1;1 and SPIol;Bsas2;1 recombinant proteins, spinach SPIol;Bsas3;1 and Arabidopsis ARAth;Bsas3;1 recombinant proteins exhibited preferred substrate specificities for the CAS reaction rather than for the CS reaction, which identified these Bsas3 isoforms as CAS. Immunoblot studies supported this conclusion. This is the first report of the identification of CAS synthase-encoding cDNAs in a living organism. A new nomenclature for CS-like proteins in plants is also proposed. PMID:10889265

  5. A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes.

    PubMed

    Welsch, Ralf; Wüst, Florian; Bär, Cornelia; Al-Babili, Salim; Beyer, Peter

    2008-05-01

    We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5' untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.

  6. A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes1[W

    PubMed Central

    Welsch, Ralf; Wüst, Florian; Bär, Cornelia; Al-Babili, Salim; Beyer, Peter

    2008-01-01

    We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5′ untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1. PMID:18326788

  7. School Lighting.

    ERIC Educational Resources Information Center

    Rennhackkamp, W. M. H.

    Research gathered by the Functional Efficiency Division of the National Building Research Institute, South Africa, is aimed at providing lighting conditions under which the school child can produce his maximum effort with the least strain and fatigue. These favorable conditions are outlined along with specific examples of their realization in…

  8. Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.).

    PubMed

    Zheng, Desen; Hrazdina, Geza

    2008-02-15

    Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3' region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product.

  9. Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

    PubMed

    Turbeville, Tracy D; Zhang, Junshun; Adams, W Christopher; Hunter, Gregory A; Ferreira, Gloria C

    2011-07-01

    5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

    PubMed Central

    Lei, Lei; Bashline, Logan; Li, Shundai

    2015-01-01

    Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress. PMID:26443667

  11. CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

    SciTech Connect

    Lei, Lei; Singh, Abhishek; Bashline, Logan; Li, Shundai; Yingling, Yaroslava G.; Gu, Ying

    2015-10-06

    Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.

  12. Ultraviolet-B light treatment increases antioxidant capacity of carrot products

    USDA-ARS?s Scientific Manuscript database

    Abiotic stresses such as cutting and ultraviolet (UV) light exposure of plant cells triggers an increased activity response by phenylalanine ammonia-lyase (PAL) and chalcone synthase resulting in increased synthesis of phenolic compounds, mainly anthocyanins and flavonoids. This study investigated ...

  13. 4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates.

    PubMed

    Zuurbier, K W; Leser, J; Berger, T; Hofte, A J; Schröder, G; Verpoorte, R; Schröder, J

    1998-12-01

    Valerophenone synthase (VPS) is a polyketide synthase that catalyzes the formation of the phloroglucinol derivatives in the synthesis of the bitter acids in hop (Humulus lupulus). The reaction uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that of the chalcone synthase in flavonoid biosynthesis. Our study showed that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone derivatives, not the products expected from three condensation reactions. The results are discussed in the context of the functional diversity and evolution in the family of CHS-related polyketide synthases.

  14. A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T.

    PubMed

    Krieger, C J; Roseboom, W; Albracht, S P; Spormann, A M

    2001-04-20

    The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.

  15. No-Light Light Bulbs

    ERIC Educational Resources Information Center

    Modern Schools, 1976

    1976-01-01

    A thumbnail sketch of some of the light bulbs manufactured for a purpose other than seeing. These "dark" lamps perform varied tasks including keeping food fresh, detecting and preventing disease, spurring plant growth, heating, and copying printed material. (Author/MLF)

  16. No-Light Light Bulbs

    ERIC Educational Resources Information Center

    Modern Schools, 1976

    1976-01-01

    A thumbnail sketch of some of the light bulbs manufactured for a purpose other than seeing. These "dark" lamps perform varied tasks including keeping food fresh, detecting and preventing disease, spurring plant growth, heating, and copying printed material. (Author/MLF)

  17. Purification, Structure and Properties of Escherichia coli tRNA Pseudouridine Synthase 1.

    DTIC Science & Technology

    1987-01-01

    RD-8193 9" PURIFICATION STRUCTURE AMD PROPERTIES OF ESCNERICHIA 11 COLI TRt4A PSEUDOURIDINE SYNTHASE 1(U) CALIFORNIA UNY OAKLAND NAVAL BIOSCIENCES...Keywo rd S: tN Pseudou ridine Synthase 1, Escherichia Cal i, 03 Plasmid, 19. ABSTRACT (Continue on reverse if necessary and identify by block number...The RNA modification enzyme, tRNA pseudouridine synthase I (PSUI) has been isolated in 95% purity from an Escherichia coli strain harboring a

  18. Light Sources and Lighting Circuits

    NASA Astrophysics Data System (ADS)

    Honda, Hisashi; Suwa, Takumi; Yasuda, Takeo; Ohtani, Yoshihiko; Maehara, Akiyoshi; Okada, Atsunori; Komatsu, Naoki; Mannami, Tomoaki

    According to the Machinery Statistics of the Ministry of Economy, Trade and Industry, the production of incandescent lamps in Japan in 2007 was 990 million units (90.0% of the previous year's total), in which the production of incandescent lamps for general lighting was 110 million units (90.0% of the previous year's total) and of tungsten-halogen lamps was 44 million units (96.6% of the previous year's total). The production of fluorescent lamps was 927 million units (93.9% of the previous year's total), in which general fluorescent lamps, excluding those for LCD back lighting, was 320 million units (87.2% of the previous year's total). Also, the production of HID lamps was 10 million units (101.5% of the previous year's total). On the other hand, when the numbers of sales are compared with the sales of the previous year, incandescent lamps for general use was 99.8%, tungsten-halogen lamps was 96.9%, fluorescent lamps was 95.9%, and HID lamps was 98.9%. Self-ballasted fluorescent lamps alone showed an increase in sales as strong as 29 million units, or 121.7% of the previous year's sales. It is considered that the switchover of incandescent lamps to HID lamps was promoted for energy conservation and carbon dioxide reduction with the problem of global warming in the background. In regard to exhibitions, Lighting Fair 2007 was held in Tokyo in March, and LIGHTFAIR INTERNATIONAL 2007 was held in New York in May. Regarding academic conferences, LS:11 (the 11th International Symposium on the Science & Technology of Light Sources) was held in Shanghai in May, and the First International Conference on White LEDs and Solid State Lighting was held in Tokyo in November. Both conferences suggested that there are strong needs and concerns now about energy conservation, saving natural resources, and restrictions of hazardous materials. In regard to incandescent lamps, the development of products aiming at higher efficacy, electric power savings, and longer life was advanced by

  19. Glareless light-emitting diode lighting tube

    NASA Astrophysics Data System (ADS)

    Chang, Rong-Seng; Li, Tung-Yen; Jwo, Ko-Wen; Wang, Sha-Wei; Tsai, Jang-Zern

    2012-03-01

    We develop a novel light bar waveguide design to produce a glareless light-emitting diode (LED) lighting tube. We design optimal parameters, such as the gap y between the tube and the reflective surface, the relative distance x between the lens and the LED, and so on. Using these parameters, we fabricate an illumination system consisting of LED light bulb installed at both ends of lighting tube. The lighting tube is shaped the same as a traditional fluorescent lighting tube in order to replace traditional lighting tubes without the modification of the lighting stand. The LED lighting tube is glareless to the observer from the side view.

  20. EDITORIAL: Controlling light with light

    NASA Astrophysics Data System (ADS)

    Hesselink, Lambertus; Feinberg, Jack; Roosen, Gerald

    2008-11-01

    The field of photorefractive physics and optics is mature and, although there is no significant commercial activity using photorefractive media, researchers in the field have had an extraordinary impact on many related areas of research and development. For example, in the late 1990s many of the telecom innovations and products were based on the interaction between light and matter. Examples include optical switches, filters, gratings, routers and light sources. The theory of multiple interacting beams of light inside a photosensitive medium, many of which were developed or further explored in photorefractive media, has found application in medicine, engineering, communication systems, displays and other photonics devices. On the occasions of the 30th anniversary of the theory of coupled wave analysis and the 10th anniversary of the meetings on Photorefractive Effects and Devices, it seemed appropriate to the meeting organizers of PR'07 to broaden the scope to include other related fields. The name of the meeting was changed to Controlling Light with Light: Photorefractive Effects, Photosensitivity, Fiber Gratings, Photonic Materials and More to attract a larger audience than traditionally would attend the more narrowly focused photorefractive meeting. To further disseminate the results of the 2007 meeting, Gerald Roosen proposed a special publication of original full research articles arising from key presentations at the meeting. The selection of papers in this Cluster Issue of Journal of Physics D: Applied Physics is the result of that initiative. We would like to thank all the authors for their contributions, the committee members for their valuable insight and efforts in helping to organize the meeting, and the Optical Society of America for their professional assistance throughout the preparation period of the meeting as well as during the three beautiful days in Lake Tahoe, CA.

  1. Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation 1

    PubMed Central

    Huber, Joan L.; Hite, Daniel R. C.; Outlaw, William H.; Huber, Steven C.

    1991-01-01

    Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with [γ-32P]ATP (JLA Huber, SC Huber, TH Nielsen [1989] Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25°C before assay. The “spontaneous” inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg2+ and was relatively insensitive to Ca2+ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32P]Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25°C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo. PMID:16667968

  2. Functional regulation of neuronal nitric oxide synthase expression and activity in the rat retina.

    PubMed

    Walter, Lais Takata; Higa, Guilherme Shigueto Vilar; Schmeltzer, Christian; Sousa, Erica; Kinjo, Erika Reime; Rüdiger, Sten; Hamassaki, Dânia Emi; Cerchiaro, Giselle; Kihara, Alexandre Hiroaki

    2014-11-01

    In the nervous system within physiological conditions, nitric oxide (NO) production depends on the activity of nitric oxide synthases (NOSs), and particularly on the expression of the neuronal isoform (nNOS). In the sensory systems, the role of NO is poorly understood. In this study, we identified nNOS-positive cells in the inner nuclear layer (INL) of the rat retina, with distinct characteristics such as somata size, immunolabeling level and location. Employing mathematical cluster analysis, we determined that nNOS amacrine cells are formed by two distinct populations. We next investigated the molecular identity of these cells, which did not show colocalization with calbindin (CB), choline acetyltransferase (ChAT), parvalbumin (PV) or protein kinase C (PKC), and only partial colocalization with calretinin (CR), revealing the accumulation of nNOS in specific amacrine cell populations. To access the functional, circuitry-related roles of these cells, we performed experiments after adaptation to different ambient light conditions. After 24h of dark-adaptation, we detected a subtle, yet statistically significant decrease in nNOS transcript levels, which returned to steady-state levels after 24h of normal light-dark cycle, revealing that nNOS expression is governed by ambient light conditions. Employing electron paramagnetic resonance (EPR), we demonstrated that dark-adaptation decreases NO production in the retina. Furthermore, nNOS accumulation changed in the dark-adapted retinas, with a general reduction in the inner plexiform layer. Finally, computational analysis based on clustering techniques revealed that dark-adaptation differently affected both types of nNOS-positive amacrine cells. Taken together, our data disclosed functional regulation of nNOS expression and activity, disclosing new circuitry-related roles of nNOS-positive cells. More importantly, this study indicated unsuspected roles for NO in the sensory systems, particularly related to adaptation to

  3. Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha[OPEN

    PubMed Central

    Zhuang, Xun; Jiang, Zuodong; Jia, Qidong; Babbitt, Patricia C.

    2016-01-01

    Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases. PMID:27650333

  4. Effect of ions of potassium and lithium on NO synthase expression in the human adrenal cortex.

    PubMed

    Kovzun, E I; Lukashenya, O S; Pushkarev, V M; Mikosha, A S; Tron'ko, N D

    2014-01-01

    The expression of endothelial and inducible NO synthase in the human adrenal glands was studied under a change in the concentration of K(+), which plays a regulatory role in aldosterone secretion. K(+) ions stimulated the expression of both isoforms of NO synthase in the human adrenal cortex. A stimulatory effect of K(+) on NO synthase is probably related to activation of the calmodulin system and potassium-induced translocation of protein kinase C. Lithium produced n inhibitory effect on both isoforms of NO synthase, which suggests that protein kinase C serves a major regulator of expression in the human adrenal glands.

  5. The type I fatty acid and polyketide synthases: a tale of two megasynthases

    PubMed Central

    Tsai, Shiou-Chuan

    2008-01-01

    This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

  6. Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism.

    PubMed

    Jia, Y; Yuan, W; Wodzinska, J; Park, C; Sinskey, A J; Stubbe, J

    2001-01-30

    The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.

  7. Restricted Role for Methionine Synthase Reductase Defined by Subcellular Localization

    PubMed Central

    Froese, DS; Wu, X; Zhang, J; Dumas, R; Schoel, WM; Amrein, M; Gravel, RA

    2009-01-01

    Methionine synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of methionine synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target to the cytosol where methionine synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target to mitochondria was also suggested by the work of Leal et al. (2004) who showed that it can act as the reducing enzyme in combination with MMAB (ATP:cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin. PMID:18221906

  8. A Critical Role for Ceramide Synthase 2 in Liver Homeostasis

    PubMed Central

    Pewzner-Jung, Yael; Park, Hyejung; Laviad, Elad L.; Silva, Liana C.; Lahiri, Sujoy; Stiban, Johnny; Erez-Roman, Racheli; Brügger, Britta; Sachsenheimer, Timo; Wieland, Felix; Prieto, Manuel; Merrill, Alfred H.; Futerman, Anthony H.

    2010-01-01

    Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22–C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis. PMID:20110363

  9. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems

    PubMed Central

    2015-01-01

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75–106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain–domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology. PMID:24779441

  10. Assembly line polyketide synthases: mechanistic insights and unsolved problems.

    PubMed

    Khosla, Chaitan; Herschlag, Daniel; Cane, David E; Walsh, Christopher T

    2014-05-13

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75-106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain-domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.

  11. Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.

    PubMed Central

    Ha, S B; Smith, A P; Howden, R; Dietrich, W M; Bugg, S; O'Connell, M J; Goldsbrough, P B; Cobbett, C S

    1999-01-01

    Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species. PMID:10368185

  12. Synthesis of antifungal glucan synthase inhibitors from enfumafungin.

    PubMed

    Zhong, Yong-Li; Gauthier, Donald R; Shi, Yao-Jun; McLaughlin, Mark; Chung, John Y L; Dagneau, Philippe; Marcune, Benjamin; Krska, Shane W; Ball, Richard G; Reamer, Robert A; Yasuda, Nobuyoshi

    2012-04-06

    An efficient, new, and scalable semisynthesis of glucan synthase inhibitors 1 and 2 from the fermentation product enfumafungin 3 is described. The highlights of the synthesis include a high-yielding ether bond-forming reaction between a bulky sulfamidate 17 and alcohol 4 and a remarkably chemoselective, improved palladium(II)-mediated Corey-Yu allylic oxidation at the highly congested C-12 position of the enfumafungin core. Multi-hundred gram quantities of the target drug candidates 1 and 2 were prepared, in 12 linear steps with 25% isolated yield and 13 linear steps with 22% isolated yield, respectively.

  13. Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus

    PubMed Central

    Lartigue, Audrey; Jeudy, Sandra; Bertaux, Lionel; Abergel, Chantal

    2013-01-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF. PMID:23295487

  14. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    PubMed

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  15. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    PubMed

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. © 2014 Wiley Periodicals, Inc.

  16. Producing a trimethylpentanoic acid using hybrid polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2014-10-07

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

  17. Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor

    PubMed Central

    Spoonamore, James E.; Roberts, Sue A.; Heroux, Annie; Bandarian, Vahe

    2008-01-01

    The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate. PMID:18931427

  18. Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins

    PubMed Central

    Balakrishnan, Nataraj; Ganesan, Sadhasivam; Rajasekaran, Padma; Rajendran, Lingeshwaran; Teddu, Sivaprasad

    2016-01-01

    ABSTRACT Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher kcat/Km values. The efficacy and industrial scalability of one of the selected variants, CefFGOS, were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS. IMPORTANCE 7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme

  19. Glycogen synthase kinase 3 in the world of cell migration.

    PubMed

    Sun, Tong; Rodriguez, Marbelys; Kim, Leung

    2009-12-01

    Glycogen synthase kinase 3 (GSK3) is one of the few master switch kinases that regulate many aspects of cell functions. Recent studies on cell polarization and migration have shown that GSK3 is also essential for proper regulation of these processes. GSK3 influences cell migration as one of the regulators of the spatiotemporally controlled dynamics of the actin cytoskeleton, microtubules, and cell-to-matrix adhesions. In this mini-review, the effects of GSK3 on these three aspects of cell migration will be discussed.

  20. Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase.

    PubMed

    Girish, Tavarekere S; Sharma, Eshita; Gopal, B

    2008-08-20

    Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.

  1. The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

    PubMed Central

    Schönitzer, Veronika; Weiss, Ingrid M

    2007-01-01

    Background Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM – 10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types. PMID:17986326

  2. Expression of a GALACTINOL SYNTHASE gene is positively associated with desiccation tolerance of Brassica napus seeds during development.

    PubMed

    Li, Xu; Zhuo, Jiajin; Jing, Yin; Liu, Xiao; Wang, Xiaofeng

    2011-10-15

    Desiccation tolerance of seeds is positively correlated with raffinose family oligosaccharides (RFOs). However, RFOs' role in desiccation tolerance is still a matter of controversy. The aim of this work was to monitor the accumulation of RFO during acquisition of desiccation tolerance in rapeseed (Brassica napus L.). Rapeseeds become desiccation tolerant at 21-24d after flowering (DAF), and the time was coincident with an accumulation of raffinose and stachyose. A gene encoding galactinol synthase (GolS; EC2.4.1.123), involved in RFO biosynthesis, was cloned and functionally characterized. Enzymatic properties of recombinant galactinol synthase were also determined. Accumulation of BnGOLS-1 mRNA in developing rapeseeds was concomitant with dry weight deposition and the acquisition of desiccation tolerance, and was concurrent with the formation of raffinose and stachyose. The physiological implications of BnGOLS-1 expression patterns in developing seeds are discussed in light of the hypothesized role of RFOs in seed desiccation tolerance. Copyright © 2011 Elsevier GmbH. All rights reserved.

  3. A monogalactosyldiacylglycerol synthase found in the green sulfur bacterium Chlorobaculum tepidum reveals important roles for galactolipids in photosynthesis.

    PubMed

    Masuda, Shinji; Harada, Jiro; Yokono, Makio; Yuzawa, Yuichi; Shimojima, Mie; Murofushi, Kazuhiro; Tanaka, Hironori; Masuda, Hanako; Murakawa, Masato; Haraguchi, Tsuyoshi; Kondo, Maki; Nishimura, Mikio; Yuasa, Hideya; Noguchi, Masato; Oh-Oka, Hirozo; Tanaka, Ayumi; Tamiaki, Hitoshi; Ohta, Hiroyuki

    2011-07-01

    Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.

  4. Light fantastic

    NASA Astrophysics Data System (ADS)

    2010-05-01

    The laser has become so ubiquitous that it would be impossible to acknowledge everyone who has played a role in its success. As Roy Glauber said at the 2005 Nobel-prize banquet, when it comes to lasers, "many hands make light work". And he should know: the prize Glauber shared with fellow optics pioneers John Hall and Theodore Hänsch is one of more than 10 Nobels awarded (so far!) for laser-related research. This timeline marking 50 years of the laser contains Physics World's pick of events from laser history, including prizes (gold text), applications (green) and "firsts" (blue).

  5. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    PubMed

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.

  6. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    PubMed

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  7. Fine structure analysis of Salmonella typhimurium glutamate synthase genes.

    PubMed Central

    Madonna, M J; Fuchs, R L; Brenchley, J E

    1985-01-01

    Glutamate synthase activity is required for the growth of Salmonella typhimurium on media containing a growth-rate-limiting nitrogen source. Mutations that alter glutamate synthase activity had been identified in the gltB gene, but it was not known which of the two nonidentical subunits of the enzyme was altered. To examine the gene-protein relationship of the glt region, two nonsense mutations were identified and used to demonstrate that gltB encodes the large subunit of the enzyme. Six strains with independent Mu cts d1 (lac bla) insertions were isolated, from which a collection of deletion mutations was obtained. The deletions were transduced with the nonsense mutations and 38 other glt point mutations to construct a fine-structure genetic map. Chromosome mobilization studies, mediated by Hfr derivatives of Mu cts d1 lysogens, showed that gltB is transcribed in a clockwise direction, as shown in the S. typhimurium linkage map. Studies of the polar effects of three Mu cts d1 insertions indicated that the gene for the small subunit maps clockwise to gltB and that the two genes are cotranscribed to form a glt operon. Images PMID:3881392

  8. An uncultivated crenarchaeota contains functional bacteriochlorophyll a synthase.

    PubMed

    Meng, Jun; Wang, Fengping; Wang, Feng; Zheng, Yanping; Peng, Xiaotong; Zhou, Huaiyang; Xiao, Xiang

    2009-01-01

    A fosmid clone 37F10 containing an archaeal 16S rRNA gene was screened out from a metagenomic library of Pearl River sediment, southern China. Sequence analysis of the 35 kb inserted fragment of 37F10 found that it contains a single 16S rRNA gene belonging to Miscellaneous Crenarchaeotal Group (MCG) and 36 open reading frames (ORFs). One ORF (orf11) encodes putative bacteriochlorophyll a synthase (bchG) gene. Bacteriochlorophyll a synthase gene has never been reported in a member of the domain Archaea, in accordance with the fact that no (bacterio)-chlorophyll has ever been detected in any cultivated archaea. The putative archaeal bchG (named as ar-bchG) was cloned and heterologously expressed in Escherichia coli. The protein was found to be capable of synthesizing bacteriochlorophyll a by esterification of bacteriochlorophyllide a with phytyl diphosphate or geranylgeranyl diphosphate. Furthermore, phylogenetic analysis clearly indicates that the ar-bchG diverges before the bacterial bchGs. Our results for the first time demonstrate that a key and functional enzyme for bacteriochlorophyll a biosynthesis does exist in Archaea.

  9. Ack kinase regulates CTP synthase filaments during Drosophila oogenesis.

    PubMed

    Strochlic, Todd I; Stavrides, Kevin P; Thomas, Sam V; Nicolas, Emmanuelle; O'Reilly, Alana M; Peterson, Jeffrey R

    2014-11-01

    The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.

  10. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    PubMed

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  11. SBSPKS: structure based sequence analysis of polyketide synthases

    PubMed Central

    Anand, Swadha; Prasad, M. V. R.; Yadav, Gitanjali; Kumar, Narendra; Shehara, Jyoti; Ansari, Md. Zeeshan; Mohanty, Debasisa

    2010-01-01

    Polyketide synthases (PKSs) catalyze biosynthesis of a diverse family of pharmaceutically important secondary metabolites. Bioinformatics analysis of sequence and structural features of PKS proteins plays a crucial role in discovery of new natural products by genome mining, as well as in design of novel secondary metabolites by biosynthetic engineering. The availability of the crystal structures of various PKS catalytic and docking domains, and mammalian fatty acid synthase module prompted us to develop SBSPKS software which consists of three major components. Model_3D_PKS can be used for modeling, visualization and analysis of 3D structure of individual PKS catalytic domains, dimeric structures for complete PKS modules and prediction of substrate specificity. Dock_Dom_Anal identifies the key interacting residue pairs in inter-subunit interfaces based on alignment of inter-polypeptide linker sequences to the docking domain structure. In case of modular PKS with multiple open reading frames (ORFs), it can predict the cognate order of substrate channeling based on combinatorial evaluation of all possible interface contacts. NRPS–PKS provides user friendly tools for identifying various catalytic domains in the sequence of a Type I PKS protein and comparing them with experimentally characterized PKS/NRPS clusters cataloged in the backend databases of SBSPKS. SBSPKS is available at http://www.nii.ac.in/sbspks.html. PMID:20444870

  12. Cryptic polyketide synthase genes in non-pathogenic Clostridium SPP.

    PubMed

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides.

  13. In vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    PubMed Central

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian; Krucewicz, Katarzyna; Beeren, Sophie R.; Rydhal, Maja G.; Yoshimura, Yayoi; Striebeck, Alexander; Motawia, Mohammed S.; Willats, William G. T.; Palcic, Monica M.

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates. PMID:26858729

  14. Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene.

    PubMed

    Shaik, Abjal Pasha; Alsaeed, Abbas H; Sultana, Asma

    2012-01-01

    The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther's disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref NP_000366.1, NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14(th) Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.

  15. Diagnosis of cystathionine beta-synthase deficiency by genetic analysis.

    PubMed

    Suri, Fatemeh; Narooie-Nejad, Mehrnaz; Safari, Iman; Moazzeni, Hamidreza; Rohani, Mohammad-Reza; Khajeh, Ali; Klotzle, Brandy; Fan, Jian-Bing; Elahi, Elahe

    2014-12-15

    Intellectual disability like other common diseases is often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. We present diagnosis of cystathionine beta-synthase (CBS) deficiency in a multiply affected Iranian family with obvious intellectual disability based on whole genome SNP homozygosity mapping. Diagnosis based on clinical presentations had not been made because of unavailability of appropriate medical services. Genetic analysis led to identification of homozygous c.346G>A in CBS that causes p.Gly116Arg in the encoded protein, cystathionine beta-synthase. CBS is the most common causative gene of homocystinurea. Later, the same mutation was found in three other apparently unrelated Iranian homocystinuria patients. p.Gly116Arg was reported once before in a Turkish patient, suggesting it may be a common CBS deficiency causing mutation in the Middle East. Clinical features of the patients are reported that evidence to variable presentations caused by the same mutation. Finally, observations in heterozygous carriers of the mutation suggest data that a single allele of the p.Gly116Arg causing mutation may have phenotypic consequences, including cardiac related phenotypes. Our study attests to the powers of genetic analysis for diagnosis especially for some forms of intellectual disability, with known genetic causing agents. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. IPC synthase as a useful target for antifungal drugs.

    PubMed

    Sugimoto, Yuichi; Sakoh, Hiroki; Yamada, Koji

    2004-12-01

    Inositol phosphorylceramide (IPC) synthase is a common and essential enzyme in fungi and plants, which catalyzes the transfer of phosphoinositol to the C-1 hydroxy of ceramide to produce IPC. This reaction is a key step in fungal sphingolipid biosynthesis, therefore the enzyme is a potential target for the development of nontoxic therapeutic antifungal agents. Natural products with a desired biological activity, aureobasidin A (AbA), khafrefungin, and galbonolide A, have been reported. AbA, a cyclic depsipeptide containing 8 amino acids and a hydroxyl acid, is a broad spectrum antifungal with strong activity against many pathogenic fungi such as Candida spp., Cryptococcus neoformans, and some Aspergillus spp. Khafrefungin, an aldonic acid ester with a C22 long alkyl chain, has antifungal activity against C. albicans, Cr. Neoformans, and Saccharomyces cerevisiae. Galbonolide A is a 14-membered macrolide with fungicidal activity against clinically important strains, and is especially potent against Cr. neoformans. These classes of natural products are potent and specific antifungal agents. We review current progress in the development of IPC synthase inhibitors with antifungal activities, and present structure-activity relationships (SAR), physicochemical and structural properties, and synthetic methodology for chemical modification.

  17. Stereochemical course of enzyme-catalyzed aminopropyl transfer: spermidine synthase

    SciTech Connect

    Kullberg, D.W.; Orr, G.R.; Coward, J.K.

    1986-05-01

    The R and S enantionmers of S-adenosyl-3-(/sup 2/H)3-(methylthio)-1-propylamine (decarboxylated S-adenosylmethionine), previously synthesized in this laboratory, were incubated with (1,4-/sup 2/H/sub 4/)-putrescine in the presence of spermidine synthase from E. coli. The resulting chiral (/sup 2/H/sub 5/)spermidines were isolated and converted to their N/sub 1/,N/sub 7/-dibocspermidine-N/sub 4/-(1S,4R)-camphanamides. The derivatives were analyzed by 500 MHz /sup 1/H-NMR and the configuration of the chiral center assigned by correlation with the spectra of synthetic chiral (/sup 2/H/sub 3/)dibocspermidine camphanamide standards. The enzyme-catalyzed aminopropyl transfer was shown to occur with net retention of configuration, indicative of a double-displacement mechanism. This result concurs with that of a previous steady-state kinetics study of spermidine synthase isolated from E. coli, but contradicts the single-displacement mechanism suggested by a stereochemical analysis of chiral spermidines biosynthesized in E. coli treated with chirally deuterated methionines. It also indicates that this aminopropyltransferase is mechanistically distinct from the methyltransferases, which have been shown to act via a single-displacement mechanism (net inversion at -CH/sub 3/) in all cases studied to date.

  18. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    PubMed

    Banzhaf, Manuel; van den Berg van Saparoea, Bart; Terrak, Mohammed; Fraipont, Claudine; Egan, Alexander; Philippe, Jules; Zapun, André; Breukink, Eefjan; Nguyen-Distèche, Martine; den Blaauwen, Tanneke; Vollmer, Waldemar

    2012-07-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.

  19. Inducible nitric oxide synthase as a possible target in hypertension.

    PubMed

    Oliveira-Paula, Gustavo H; Lacchini, Riccardo; Tanus-Santos, Jose E

    2014-02-01

    Nitric oxide (NO) is an important vasodilator produced by vascular endothelium. Its enzymatic formation is derived from three different synthases: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) synthases. While relatively small amounts of NO produced by eNOS are important to cardiovascular homeostasis, high NO levels produced associated with iNOS activity may have detrimental consequences to the cardiovascular system and contribute to hypertension. In this article, we reviewed current literature and found mounting evidence indicating that increased iNOS expression and activity contribute to the pathogenesis of hypertension and its complications. Excessive amounts of NO produced by iNOS up-regulation can react with superoxide anions forming peroxynitrite, thereby promoting nitrosative stress and endothelial dysfunction. In addition, abnormal iNOS activity can up-regulate arginase activity, allowing it to compete with eNOS for L-arginine, thereby resulting in reduced NO bioavailability. This may also lead to eNOS uncoupling with enhanced production of superoxide anions instead of NO. All these alterations mediated by iNOS apparently contribute to hypertension and its complications. We also reviewed current evidence showing the effects of iNOS inhibitors on different animal models of hypertension. iNOS inhibition apparently exerts antihypertensive effects, decreases oxidative and nitrosative stress, and improves vascular function. Together, these studies highlight the possibility that iNOS is a potential pharmacological target in hypertension.

  20. Trimethylguanosine Synthase1 (TGS1) Is Essential for Chilling Tolerance.

    PubMed

    Gao, Jinpeng; Wallis, James G; Jewell, Jeremy B; Browse, John

    2017-07-01

    Chilling stress is a major factor limiting plant development and crop productivity. Because the plant response to chilling is so complex, we are far from understanding the genes important in the response to chilling. To identify new genes important in chilling tolerance, we conducted a novel mutant screen, combining a confirmed SALK T-DNA insertion collection with traditional forward genetics. We screened a pool of more than 3700 confirmed homozygous SALK T-DNA insertion lines for visible defects under prolonged growth at 5°C. Of the chilling-sensitive mutants we observed, mutations at one locus were characterized in detail. This gene, At1g45231, encodes an Arabidopsis (Arabidopsis thaliana) trimethylguanosine synthase (TGS1), previously uncharacterized in the plant kingdom. We confirmed that Arabidopsis TGS1 is a functional ortholog of other trimethylguanosine synthases based both on its in vitro methyltransferase activity and on its ability to rescue the cold-growth inhibition of a Saccharomyces cerevisiae tgs1Δ mutant in vivo. While tgs1 mutant plants grew normally at 22°C, their vegetative and reproductive growth was severely compromised under chilling conditions. When we transgenically expressed TGS1 in the mutant plants, the chilling-sensitive phenotype was relieved, demonstrating that TGS1 is required for chilling tolerance. © 2017 American Society of Plant Biologists. All Rights Reserved.

  1. [Cloning, expression and charaterization of chalcone synthase from Saussurea medusa].

    PubMed

    Xia, Fang; Li, Houhua; Fu, Chunxiang; Yu, Zhenzhen; Xu, Yanjun; Zhao, Dexiu

    2011-09-01

    A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1313 bp, containing an open reading frame (1170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

  2. Chromosomal localization of the human and mouse hyaluronan synthase genes

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Seldin, M.F.

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  3. Tryptophan synthase: a multienzyme complex with an intramolecular tunnel.

    PubMed

    Miles, E W

    2001-01-01

    Tryptophan synthase is a classic enzyme that channels a metabolic intermediate, indole. The crystal structure of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium revealed for the first time the architecture of a multienzyme complex and the presence of an intramolecular tunnel. This remarkable hydrophobic tunnel provides a likely passageway for indole from the active site of the alpha subunit, where it is produced, to the active site of the beta subunit, where it reacts with L-serine to form L-tryptophan in a pyridoxal phosphate-dependent reaction. Rapid kinetic studies of the wild type enzyme and of channel-impaired mutant enzymes provide strong evidence for the proposed channeling mechanism. Structures of a series of enzyme-substrate intermediates at the alpha and beta active sites are elucidating enzyme mechanisms and dynamics. These structural results are providing a fascinating picture of loops opening and closing, of domain movements, and of conformational changes in the indole tunnel. Solution studies provide further evidence for ligand-induced conformational changes that send signals between the alpha and beta subunits. The combined results show that the switching of the enzyme between open and closed conformations couples the catalytic reactions at the alpha and beta active sites and prevents the escape of indole.

  4. Eugenol synthase genes in floral scent variation in Gymnadenia species.

    PubMed

    Gupta, Alok K; Schauvinhold, Ines; Pichersky, Eran; Schiestl, Florian P

    2014-12-01

    Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

  5. The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression

    PubMed Central

    Rabender, Christopher S.; Alam, Asim; Sundaresan, Gobalakrishnan; Cardnell, Robert J.; Yakovlev, Vasily A.; Mukhopadhyay, Nitai D.; Graves, Paul; Zweit, Jamal; Mikkelsen, Ross B.

    2015-01-01

    Here evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast to normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on HPLC analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin: dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid and head and neck tumors compared to normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling including increased cGMP-dependent protein kinase (PKG) activity, decreased β-catenin expression and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by clonogenic assay, Ki67 staining and 18F-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and as a consequence nitric oxide synthase activity generates more peroxynitrite and superoxide anion than nitric oxide resulting in important tumor growth promoting and anti-apoptotic signaling properties. Implications The synthetic BH4, Kuvan®, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth. PMID:25724429

  6. The crystal structure of human GDP-L-fucose synthase.

    PubMed

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  7. From bacterial to human dihydrouridine synthase: automated structure determination

    PubMed Central

    Whelan, Fiona; Jenkins, Huw T.; Griffiths, Samuel C.; Byrne, Robert T.; Dodson, Eleanor J.; Antson, Alfred A.

    2015-01-01

    The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr_rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer. PMID:26143927

  8. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

    PubMed

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C

    2012-04-27

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Cutaneous dyspigmentation in patients with ganglioside GM3 synthase deficiency.

    PubMed

    Wang, Heng; Bright, Alicia; Xin, Baozhong; Bockoven, J R; Paller, Amy S

    2013-04-01

    Ganglioside GM3 synthase deficiency is a rare autosomal recessive metabolic disorder characterized by infantile onset of severe irritability and epilepsy, failure to thrive, developmental stagnation, and cortical blindness. Because of the lack of easily recognizable dysmorphism and specific neurologic manifestations, identification of patients with this condition is extremely challenging. Here we report on previously undescribed pigmentary abnormalities in 20 of 38 patients with GM3 synthase deficiency. All 20 of the patients showed freckle-like hyperpigmented macules, ranging in size from 2 to 5 mm in diameter and usually found bilaterally on the extremities, especially the dorsal aspects of the hands and feet. Seven of these patients also had depigmented macules and patches, especially on the face and extremities. These cutaneous changes were asymptomatic, and were not associated with the severity or particular phenotype of the neurologic disease. They became visible only after the first years of life with an increased incidence with advancing age. These distinct pigmentary features are not identified in 54 normal siblings, and may provide a useful clue in identifying patients with ganglioside metabolic disorders. Copyright © 2013 Wiley Periodicals, Inc.

  10. Farnesyl pyrophosphate synthase modulators: a patent review (2006 - 2010)

    PubMed Central

    Sun, Shuting; McKenna, Charles E.

    2012-01-01

    Introduction Farnesyl pyrophosphosphate synthase (FPPS (also known as farnesyl diphosphate synthase, FDPS)) is one of the key enzymes involved in the mevalonate pathway and as such is widely expressed. FPPS modulators, specifically FPPS inhibitors, are useful in treating a number of diseases, including bone related disorders characterized by excessive bone resorption e.g. osteoporosis, cancer metathesis to bone and infectious diseases caused by certain parasites. Areas covered This review covers structures and applications of novel FPPS modulators described in the patent literature from 2006 to 2010. Patents disclosing new formulations and uses of existing FPPS inhibitors are also reviewed. Thirty-three patents retrieved from the USPTO, EP and WIPO databases are examined with the goal of defining current trends in drug discovery related to FPPS inhibition, and its therapeutic effects. Expert opinion Bisphosphonates continue to dominate in this area, although other types of modulator are making their appearance. Remarkable for their high bone mineral affinity, bisphosphonates are structural mimics of the dimethylallyl pyrophosphate (DMAPP) substrate of FPPS, and constitute the major type of FPPS inhibitor currently used in the clinic for treatment of bone-related diseases. Lipophilic bisphosphonates and new classes of non-bisphosphonate FPPS inhibitors (salicylic acid and quinoline derivatives) have been introduced as possible alternatives for treatment of soft tissue diseases, such as some cancers. Novel formulations, fluorescent diagnostic probes and new therapeutic applications of existing FPPS inhibitors are also areas of significant patent activity, demonstrating growing recognition of the versatility and underdeveloped potential of these drugs. PMID:21702715

  11. Manipulation of pulmonary prostacyclin synthase expression prevents murine lung cancer.

    PubMed

    Keith, Robert L; Miller, York E; Hoshikawa, Yasushi; Moore, Mark D; Gesell, Tracy L; Gao, Bifeng; Malkinson, Alvin M; Golpon, Heiko A; Nemenoff, Raphael A; Geraci, Mark W

    2002-02-01

    Inhibition of cyclooxygenase (COX) activity decreases eicosanoid production and prevents lung cancer in animal models. Prostaglandin (PG) I(2) (PGI(2), prostacyclin) is a PGH(2) metabolite with anti-inflammatory, antiproliferative, and antimetastatic properties. The instability of PGI(2) has limited its evaluation in animal models of cancer. We hypothesized that pulmonary overexpression of prostacyclin synthase may prevent the development of murine lung tumors. Transgenic mice with selective pulmonary prostacyclin synthase overexpression were exposed to two distinct carcinogenesis protocols: an initiation/promotion model and a simple carcinogen model. The transgenic mice exhibited significantly reduced lung tumor multiplicity (tumor number) in proportion to transgene expression, a dose-response effect. Moreover, the highest expressing mice demonstrated reduced tumor incidence. To investigate the mechanism for protection, we evaluated PG levels and inflammatory responses. At the time of sacrifice following one carcinogenesis model, the transgenics exhibited only an increase in 6-keto-PGF(1alpha), not a decrease in PGE(2). Thus, elevated PGI(2) levels and not decreased PGE(2) levels appear to be necessary for the chemopreventive effects. When exposed to a single dose of butylated hydroxytoluene, transgenic mice exhibited a survival advantage; however, reduction in alveolar inflammatory response was not observed. These studies demonstrate that manipulation of PG metabolism downstream from COX produces even more profound lung cancer reduction than COX inhibition alone and could be the basis for new approaches to understanding the pathogenesis and prevention of lung cancer.

  12. Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase

    SciTech Connect

    Napolitano, H. B.; Sculaccio, S. A.; Thiemann, O. H.; Oliva, G.

    2005-01-01

    X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa. Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P2{sub 1}2{sub 1}2 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 Å. The crystals diffract to a maximum resolution of 3.3 Å and a complete data set to 3.5 Å resolution was collected and analysed.

  13. Fragments of ATP synthase mediate plant perception of insect attack

    PubMed Central

    Schmelz, Eric A.; Carroll, Mark J.; LeClere, Sherry; Phipps, Stephen M.; Meredith, Julia; Chourey, Prem S.; Alborn, Hans T.; Teal, Peter E. A.

    2006-01-01

    Plants can perceive a wide range of biotic attackers and respond with targeted induced defenses. Specificity in plant non-self-recognition occurs either directly by perception of pest-derived elicitors or indirectly through resistance protein recognition of host targets that are inappropriately proteolyzed. Indirect plant perception can occur during interactions with pathogens, yet evidence for analogous events mediating the detection of insect herbivores remains elusive. Here we report indirect perception of herbivory in cowpea (Vigna unguiculata) plants attacked by fall armyworm (Spodoptera frugiperda) larvae. We isolated and identified a disulfide-bridged peptide (+ICDINGVCVDA−), termed inceptin, from S. frugiperda larval oral secretions that promotes cowpea ethylene production at 1 fmol leaf−1 and triggers increases in the defense-related phytohormones salicylic acid and jasmonic acid. Inceptins are proteolytic fragments of chloroplastic ATP synthase γ-subunit regulatory regions that mediate plant perception of herbivory through the induction of volatile, phenylpropanoid, and protease inhibitor defenses. Only S. frugiperda larvae that previously ingested chloroplastic ATP synthase γ-subunit proteins and produced inceptins significantly induced cowpea defenses after herbivory. Digestive fragments of an ancient and essential plant enzyme, inceptin functions as a potent indirect signal initiating specific plant responses to insect attack. PMID:16720701

  14. 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

  15. Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis

    PubMed Central

    Li, Yuejuan; Liang, Jiurong; Yang, Ting; Mena, Jessica Monterrosa; Huan, Caijuan; Xie, Ting; Kurkciyan, Adrianne; Liu, Ningshan; Jiang, Dianhua; Noble, Paul W.

    2016-01-01

    Dysregulated repair of lung injury often results in lung fibrosis characterized by unremitting deposition of matrix components including the glycosaminoglycan hyaluronan (HA). HA is mainly produced by hyaluronan synthases (HAS) in mesenchymal cells. We previously demonstrated that over-expression of HAS2 in mesenchymal cells in mice regulates the invasiveness of fibroblasts and promotes severe lung fibrosis. The mechanisms that control the resolution of lung fibrosis are unknown. We propose that a critical step in resolving fibrosis is the induction of senescence in fibrotic fibroblasts and hyaluronan synthase 2 may regulate this process. We found that fibrotic fibroblasts developed the characteristics of replicative senescence in culture and that HAS2 expression was dramatically down-regulated. Furthermore, down-regulation of HAS2 initiated and regulated fibroblast senescence through a p27-CDK2-SKP2 pathway. Deletion of HAS2 in mouse mesenchymal cells increased the cellular senescence of fibroblasts in bleomycin-induced mouse lung fibrosis in vivo. These data suggest that HAS2 may be a critical regulator of the fate of pulmonary fibrosis and we propose a model where over-expression of HAS2 promotes an invasive phenotype resulting in severe fibrosis and down-regulation of HAS2 promotes resolution. Targeting HAS2 to induce fibroblast senescence could be an attractive approach to resolve tissue fibrosis. PMID:26987798

  16. Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis.

    PubMed

    Li, Yuejuan; Liang, Jiurong; Yang, Ting; Monterrosa Mena, Jessica; Huan, Caijuan; Xie, Ting; Kurkciyan, Adrianne; Liu, Ningshan; Jiang, Dianhua; Noble, Paul W

    2016-09-01

    Dysregulated repair of lung injury often results in lung fibrosis characterized by unremitting deposition of matrix components including glycosaminoglycan hyaluronan (HA). HA is mainly produced by hyaluronan synthases (HAS) in mesenchymal cells. We previously demonstrated that over-expression of HAS2 in mesenchymal cells in mice regulates the invasiveness of fibroblasts and promotes severe lung fibrosis. The mechanisms that control the resolution of lung fibrosis are unknown. We propose that a critical step in resolving fibrosis is the induction of senescence in fibrotic fibroblasts and hyaluronan synthase 2 may regulate this process. We found that fibrotic fibroblasts developed the characteristics of replicative senescence in culture and that HAS2 expression was dramatically down-regulated. Furthermore, down-regulation of HAS2 initiated and regulated fibroblast senescence through a p27-CDK2-SKP2 pathway. Deletion of HAS2 in mouse mesenchymal cells increased the cellular senescence of fibroblasts in bleomycin-induced mouse lung fibrosis in vivo. These data suggest that HAS2 may be a critical regulator of the fate of pulmonary fibrosis and we propose a model where over-expression of HAS2 promotes an invasive phenotype resulting in severe fibrosis and down-regulation of HAS2 promotes resolution. Targeting HAS2 to induce fibroblast senescence could be an attractive approach to resolve tissue fibrosis. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Cloricromene inhibits the induction of nitric oxide synthase.

    PubMed

    Zingarelli, B; Carnuccio, R; Di Rosa, M

    1993-10-19

    The effect of cloricromene, a coumarin derivative, was investigated on the lipopolysaccharide-stimulated nitric oxide (NO) synthase induction in intact aortas from endotoxin shocked rats and in the murine macrophage cell line J774. Rings of thoracic aortas from lipopolysaccharide (4 mg/kg, i.v.)-shocked rats, contracted with phenylephrine, showed a progressive decrease in tone, that was of a greater magnitude than that of aortas from naive rats. Moreover, a decreased response to the constrictor effect of phenylephrine was observed in aortas from shocked rats. In vivo treatment with cloricromene (2 mg/kg, i.v.) 30 min before lipopolysaccharide administration partially prevented the loss in tone of aortic rings and improved their reactivity to phenylephrine. Murine J774 macrophages activated with lipopolysaccharide (100 ng/ml) produced significant amounts of nitrites (NO2-; 28.2 +/- 3.5 nmol/10(6) cells per 24 h). Cloricromene (2, 20 or 200 microM) added to the cells concomitantly with lipopolysaccharide inhibited NO2- production in a concentration-dependent manner. Maximum inhibition (84.0 +/- 8.0%) was observed when cloricromene (200 microM) was added to the cells 6 h before lipopolysaccharide, whereas it was ineffective when given 6 h after endotoxin. These results demonstrate that cloricromene inhibits the expression but not the activity of the inducible NO synthase.

  18. Transcriptional regulation of Bacillus subtilis citrate synthase genes.

    PubMed

    Jin, S; Sonenshein, A L

    1994-08-01

    The Bacillus subtilis citrate synthase genes citA and citZ were repressed during early exponential growth phase in nutrient broth medium and were induced as cells reached the end of exponential phase. Both genes were also induced by treatment of cells with the drug decoyinine. After induction, the steady-state level of citZ mRNA was about five times higher than that of citA mRNA. At least some of the citZ transcripts read through into the isocitrate dehydrogenase (citC) gene. Transcription from an apparent promoter site located near the 3' end of the citZ gene also contributed to expression of citC. In minimal medium, citA transcription was about 6-fold lower when glucose was the sole carbon source than it was when succinate was the carbon source. Expression of the citZ gene was repressed 2-fold by glucose and 10-fold when glucose and glutamate were present simultaneously. This latter synergistic repression is similar to the effect of glucose and glutamate on steady-state citrate synthase enzyme activity. CitR, a protein of the LysR family, appeared to be a repressor of citA but not of citZ.

  19. Structural basis for glucose-6-phosphate activation of glycogen synthase

    SciTech Connect

    Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.

    2010-11-22

    Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

  20. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    PubMed

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-02

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

    PubMed Central

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides. PMID:22235310

  2. Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase

    SciTech Connect

    Shi, Dashuang Caldovic, Ljubica; Jin, Zhongmin; Yu, Xiaolin; Qu, Qiuhao; Roth, Lauren; Morizono, Hiroki; Hathout, Yetrib; Allewell, Norma M.; Tuchman, Mendel

    2006-12-01

    Expression, crystallization and preliminary X-ray diffraction studies of a novel bifunctional N-acetylglutamate synthase/kinase from X. campestris homologous to vertebrate N-acetylglutamate synthase are reported. A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6{sub 2}22, with unit-cell parameters a = b = 134.60, c = 192.11 Å, and diffract to about 3.0 Å resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method.

  3. Suboptimal Light Conditions Influence Source-Sink Metabolism during Flowering.

    PubMed

    Christiaens, Annelies; De Keyser, Ellen; Pauwels, Els; De Riek, Jan; Gobin, Bruno; Van Labeke, Marie-Christine

    2016-01-01

    Reliance on carbohydrates during flower forcing was investigated in one early and one late flowering cultivar of azalea (Rhododendron simsii hybrids). Carbohydrate accumulation, invertase activity, and expression of a purported sucrose synthase gene (RsSUS) was monitored during flower forcing under suboptimal (natural) and optimal (supplemental light) light conditions, after a cold treatment (7°C + dark) to break flower bud dormancy. Post-production sucrose metabolism and flowering quality was also assessed. Glucose and fructose concentrations and invertase activity increased in petals during flowering, while sucrose decreased. In suboptimal light conditions RsSUS expression in leaves increased as compared to optimal light conditions, indicating that plants in suboptimal light conditions have a strong demand for carbohydrates. However, carbohydrates in leaves were markedly lower in suboptimal light conditions compared to optimal light conditions. This resulted in poor flowering of plants in suboptimal light conditions. Post-production flowering relied on the stored leaf carbon, which could be accumulated under optimal light conditions in the greenhouse. These results show that flower opening in azalea relies on carbohydrates imported from leaves and is source-limiting under suboptimal light conditions.

  4. Suboptimal Light Conditions Influence Source-Sink Metabolism during Flowering

    PubMed Central

    Christiaens, Annelies; De Keyser, Ellen; Pauwels, Els; De Riek, Jan; Gobin, Bruno; Van Labeke, Marie-Christine

    2016-01-01

    Reliance on carbohydrates during flower forcing was investigated in one early and one late flowering cultivar of azalea (Rhododendron simsii hybrids). Carbohydrate accumulation, invertase activity, and expression of a purported sucrose synthase gene (RsSUS) was monitored during flower forcing under suboptimal (natural) and optimal (supplemental light) light conditions, after a cold treatment (7°C + dark) to break flower bud dormancy. Post-production sucrose metabolism and flowering quality was also assessed. Glucose and fructose concentrations and invertase activity increased in petals during flowering, while sucrose decreased. In suboptimal light conditions RsSUS expression in leaves increased as compared to optimal light conditions, indicating that plants in suboptimal light conditions have a strong demand for carbohydrates. However, carbohydrates in leaves were markedly lower in suboptimal light conditions compared to optimal light conditions. This resulted in poor flowering of plants in suboptimal light conditions. Post-production flowering relied on the stored leaf carbon, which could be accumulated under optimal light conditions in the greenhouse. These results show that flower opening in azalea relies on carbohydrates imported from leaves and is source-limiting under suboptimal light conditions. PMID:26973689

  5. Characterization of a chitin synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines

    USDA-ARS?s Scientific Manuscript database

    Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We have identified and characterized a chitin synthase gene (CHS) from cDNA of Aphis glycines, the soybean aphid, a serious pest of soybean. The full-length cDNA of CHS in A. glyc...

  6. [The influence of inhibitors of neuronal and inducible NO-synthases on experimental hemorrhagic stroke].

    PubMed

    Krushinskiĭ, A L; Kuzenkov, V S; D'iakonova, V E; Reutov, V P

    2014-01-01

    Objectives. To study the effect of inhibitors of neuronal and inducible NO-synthase on the development of hemorrhagic stroke in rats Krushinsky-Molodkina (KM) without adaptation to hypoxia and with short-term adaptation to hypobaric hypoxia. Material and methods. Ninety rats were included in the study. Experiments with short-term adaptation to hypobaric hypoxia were performed on 48 rats. The inhibitor of inducible NO-synthase (aminoguanidine, "Sigma") or the inhibitor of neuronal NO-synthase (7-nitroindasol, "Sigma") were injected in dosage 2.5 mg/100g intraperitoneally. Results. Selective inhibitors of neuronal and inducible NO-synthase had a protective effect on stress injuries in KM rats. The inhibitor of neuronal NO-synthase was more effective than the inhibitor of inducible NO-synthase in the experiments without adaptation to hypoxia. Markedly greater protective effect was achieved by the simultaneous introduction of inhibitors of neuronal and inducible NO-synthase. The greatest protective effect in the development of stress damage in rats of KM was observed in short-term adaptation to hypobaric hypoxia with simultaneous introduction of both inhibitors. Conclusions. It can be assumed that an excessive amount of NO produced by neuronal and inducible NO-synthases during the acoustic exposure in KM rats leads to stress damage. Use of selective inhibitors reduce the excess NO synthesis and the development of audiogenic stress damage caused by hemorrhagic stroke.

  7. Cloning, Expression, and Characterization of cis-Polyprenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius

    PubMed Central

    Hemmi, Hisashi; Yamashita, Satoshi; Shimoyama, Takefumi; Nakayama, Toru; Nishino, Tokuzo

    2001-01-01

    cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme. PMID:11114943

  8. Studies on 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase using chorismate mutase inhibitors.

    PubMed

    Birck, M R; Husain, A; Sheflyan, G Y; Ganem, B; Woodard, R W

    2001-11-05

    The proposed cyclic mechanism of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.

  9. Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

    PubMed Central

    Mühleip, Alexander W.; Joos, Friederike; Wigge, Christoph; Frangakis, Achilleas S.; Kühlbrandt, Werner; Davies, Karen M.

    2016-01-01

    F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology. PMID:27402755

  10. Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.

    PubMed

    Morii, Hiroyuki; Okauchi, Tatsuo; Nomiya, Hiroki; Ogawa, Midori; Fukuda, Kazumasa; Taniguchi, Hatsumi

    2013-03-01

    We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.

  11. Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

    USDA-ARS?s Scientific Manuscript database

    In cereal seeds mutations in one or more starch synthases lead to decreased amylopectin and increased amylose content. Here, the impact of starch synthase IIa (SSIIa or SGP-1) mutations upon durum starch was investigated. A screen of durum accessions identified two lines lacking SGP-A1, the A geno...

  12. A diverse family of Type III polyketide synthases in Eucalyptus species.

    PubMed

    Rubin-Pitel, Sheryl B; Luo, Yunzi; Lee, Jung-Kul; Zhao, Huimin

    2010-08-01

    Eucalyptus species synthesize a wealth of polyketide natural products, but no relevant biosynthetic enzyme has been identified. Degenerate primers designed from conserved regions of fourteen chalcone synthase superfamily enzymes were used to isolate gene fragments from at least five different Type III polyketide synthases (PKSs) in E. camaldulensis and E. robusta.

  13. Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

    PubMed

    Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

    2012-06-01

    Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

  14. Molecular cloning of an 1-aminocyclopropane-1-carboxylate synthase from senescing carnation flower petals.

    PubMed

    Park, K Y; Drory, A; Woodson, W R

    1992-01-01

    Synthetic oligonucleotides based on the sequence of 1-aminocyclopropane-1-carboxylate (ACC) synthase from tomato were used to prime the synthesis and amplification of a 337 bp tomato ACC synthase cDNA by polymerase chain reaction (PCR). This PCR product was used to screen a cDNA library prepared from mRNA isolated from senescing carnation flower petals. Two cDNA clones were isolated which represented the same mRNA. The longer of the two clones (CARACC3) contained a 1950 bp insert with a single open reading frame of 516 amino acids encoding a protein of 58 kDa. The predicted protein from the carnation ACC synthase cDNA was 61%, 61%, 64%, and 51% identical to the deduced proteins from zucchini squash, winter squash, tomato, and apple, respectively. Genomic DNA gel blot analysis indicated the presence of at least a second gene in carnation which hybridized to CARACC3 under conditions of low stringency. ACC synthase mRNA accumulates during senescence of carnation flower petals concomitant with the increase in ethylene production and ACC synthase enzyme activity. Ethylene induced the accumulation of ACC synthase mRNA in presenescent petals. Wound-induced ethylene production in leaves was not associated with an increase in ACC synthase mRNA represented by CARACC3. These results indicate that CARACC3 represents an ACC synthase transcript involved in autocatalytic ethylene production in senescing flower petals.

  15. Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

    USDA-ARS?s Scientific Manuscript database

    Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-poly...

  16. Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples 1

    PubMed Central

    Bufler, Gebhard

    1984-01-01

    Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO2 production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO2; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene. Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO2, but not by 1.25% CO2. However, each of these CO2 concentrations reduced the rate of increase of ACC synthase activity. PMID:16663569

  17. Characterization of the cDNA and gene coding for the biotin synthase of Arabidopsis thaliana.

    PubMed Central

    Weaver, L M; Yu, F; Wurtele, E S; Nikolau, B J

    1996-01-01

    Biotin, an essential cofactor, is synthesized de novo only by plants and some microbes. An Arabidopsis thaliana expressed sequence tag that shows sequence similarity to the carboxyl end of biotin synthase from Escherichia coli was used to isolate a near-full-length cDNA. This cDNA was shown to code for the Arabidopsis biotin synthase by its ability to complement a bioB mutant of E. coli. Site-specific mutagenesis indicates that residue threonine-173, which is highly conserved in biotin synthases, is important for catalytic competence of the enzyme. The primary sequence of the Arabidopsis biotin synthase is most similar to biotin synthases from E. coli, Serratia marcescens, and Saccharomyces cerevisiae (about 50% sequence identity) and more distantly related to the Bacillus sphaericus enzyme (33% sequence identity). The primary sequence of the amino terminus of the Arabidopsis biotin synthase may represent an organelle-targeting transit peptide. The single Arabidopsis gene coding for biotin synthase, BIO2, was isolated and sequenced. The biotin synthase coding sequence is interrupted by five introns. The gene sequence upstream of the translation start site has several unusual features, including imperfect palindromes and polypyrimidine sequences, which may function in the transcriptional regulation of the BIO2 gene. PMID:8819873

  18. Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase*

    PubMed Central

    Wei, Chin-Chuan; Wang, Zhi-Qiang; Tejero, Jesús; Yang, Ya-Ping; Hemann, Craig; Hille, Russ; Stuehr, Dennis J.

    2008-01-01

    Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H4B) cofactor. During Arg hydroxylation, H4B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H4B) within NOS before further catalysis can proceed. Whereas H4B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H4B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H4B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a “through-heme” mechanism may operate for H4B radical reduction in NOS. PMID:18283102

  19. Comparative structural and computational analysis supports eighteen cellulose synthases in the plant cellulose synthesis complex

    DOE PAGES

    Nixon, B. Tracy; Mansouri, Katayoun; Singh, Abhishek; ...

    2016-06-27

    A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individualmore » lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In conclusion, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.« less

  20. Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex

    PubMed Central

    Nixon, B. Tracy; Mansouri, Katayoun; Singh, Abhishek; Du, Juan; Davis, Jonathan K.; Lee, Jung-Goo; Slabaugh, Erin; Vandavasi, Venu Gopal; O’Neill, Hugh; Roberts, Eric M.; Roberts, Alison W.; Yingling, Yaroslava G.; Haigler, Candace H.

    2016-01-01

    A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In summary, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains. PMID:27345599

  1. Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types.

    PubMed Central

    Koes, RE; Van Blokland, R; Quattrocchio, F; Van Tunen, AJ; Mol, J

    1990-01-01

    Chalcone synthase (CHS) catalyzes the first step in the biosynthesis of flavonoids that function in flower pigmentation, protection against stress, and induction of nodulation. The petunia genome contains eight complete chs genes, of which four are differentially expressed in floral tissues and UV-light-induced seedlings. The 5[prime]-flanking regions of these four chs genes were fused to the [beta]-glucuronidase (GUS) reporter gene and introduced into petunia plants by Agrobacterium-mediated transformation. We show that expression of each construct is identical to the expression of the authentic chs gene, implying that the differences in expression pattern between these chs genes are caused at least in part by their promoters. Histochemical analyses of GUS expression show that chs promoters are not only active in pigmented cell types (epidermal cells of the flower corolla and tube and [sub] epidermal cells of the flower stem) but also in a number of unpigmented cell types (mesophylic cells of the corolla, several cell types in the ovary and the seed coat). Comparison of chs-GUS expression and flavonoid accumulation patterns in anthers suggests that intercellular transport of flavonoids and enzymes occurs in this organ. Analysis of the flavonoids accumulated in tissues from mutant lines shows that only a subset of the genes that control flavonoid biosynthesis in the flower operates in the ovary and seed. This implies that (genetic) control of flavonoid biosynthesis is highly tissue specific. PMID:12354962

  2. Early divergence, broad distribution, and high diversity of animal chitin synthases.

    PubMed

    Zakrzewski, Anne-C; Weigert, Anne; Helm, Conrad; Adamski, Marcin; Adamska, Maja; Bleidorn, Christoph; Raible, Florian; Hausen, Harald

    2014-02-01

    Even though chitin is one of the most abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding new light on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. The most extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs in molluscs. Independent fusion to different kinds of myosin motor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in the morphology of many animals, the here presented CS diversification reveals many evolutionary complexities. Our findings strongly suggest a very broad and multifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest.

  3. Modeling studies with Helicobacter pylori octaprenyl pyrophosphate synthase reveal the enzymatic mechanism of trans-prenyltransferases.

    PubMed

    Zhang, Jinyong; Zhang, Xiaoli; Zhang, Rui; Wu, Chao; Guo, Ying; Mao, Xuhu; Guo, Gang; Zhang, Ying; Wang, Da-Cheng; Li, Defeng; Zou, Quanming

    2012-12-01

    Octaprenyl pyrophosphate synthase (OPPs), an enzyme belonging to the trans-prenyltransferases family, is involved in the synthesis of C40 octaprenyl pyrophosphate (OPP) by reacting farnesyl pyrophosphate (FPP) with five isopentenyl pyrophosphates (IPP). It has been reported that OPPs is essential for bacteria's normal growth and is a potential target for novel antibacterial drug design. Here we report the crystal structure of OPPs from Helicobacter pylori, determined by MAD method at 2.8 Å resolution and refined to 2.0 Å resolution. The substrate IPP was docked into HpOPPs structure and residues involved in IPP recognition were identified. The other substrate FPP, the intermediate GGPP and a nitrogen-containing bisphosphonate drug were also modeled into the structure. The resulting model shed some lights on the enzymatic mechanism, including (1) residues Arg87, Lys36 and Arg39 are essential for IPP binding; (2) residues Lys162, Lys224 and Gln197 are involved in FPP binding; (3) the second DDXXD motif may involve in FPP binding by Mg(2+) mediated interactions; (4) Leu127 is probably involved in product chain length determination in HpOPPs and (5) the intermediate products such as GGPP need a rearrange to occupy the binding site of FPP and then IPP is reloaded. Our results also indicate that the nitrogen-containing bisphosphonate drugs are potential inhibitors of FPPs and other trans-prenyltransferases aiming at blocking the binding of FPP. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Enzyme That Makes You Cry-Crystal Structure of Lachrymatory Factor Synthase from Allium cepa.

    PubMed

    Silvaroli, Josie A; Pleshinger, Matthew J; Banerjee, Surajit; Kiser, Philip D; Golczak, Marcin

    2017-07-26

    The biochemical pathway that gives onions their savor is part of the chemical warfare against microbes and animals. This defense mechanism involves formation of a volatile lachrymatory factor (LF) ((Z)-propanethial S-oxide) that causes familiar eye irritation associated with onion chopping. LF is produced in a reaction catalyzed by lachrymatory factor synthase (LFS). The principles by which LFS facilitates conversion of a sulfenic acid substrate into LF have been difficult to experimentally examine owing to the inherent substrate reactivity and lability of LF. To shed light on the mechanism of LF production in the onion, we solved crystal structures of LFS in an apo-form and in complex with a substrate analogue, crotyl alcohol. The enzyme closely resembles the helix-grip fold characteristic for plant representatives of the START (star-related lipid transfer) domain-containing protein superfamily. By comparing the structures of LFS to that of the abscisic acid receptor, PYL10, a representative of the START protein superfamily, we elucidated structural adaptations underlying the catalytic activity of LFS. We also delineated the architecture of the active site, and based on the orientation of the ligand, we propose a mechanism of catalysis that involves sequential proton transfer accompanied by formation of a carbanion intermediate. These findings reconcile chemical and biochemical information regarding thioaldehyde S-oxide formation and close a long-lasting gap in understanding of the mechanism responsible for LF production in the onion.

  5. Application of Grote-Hynes theory to the reaction catalyzed by thymidylate synthase.

    PubMed

    Kanaan, Natalia; Roca, Maite; Tuñón, Iñaki; Martí, Sergio; Moliner, Vicent

    2010-10-28

    A theoretical study of dynamic effects on the rate-limiting step of the thymidylate synthase catalyzed reaction has been carried out by means of Grote-Hynes theory, successfully predicting the values of the recrossing effects for a chemical reaction that involves the transfer of a classical light particle. The transmission coefficients, obtained at 278, 293, 303, and 313 K, are almost invariant and in all cases far from unity, revealing a significant coupling of the environment motions and the reaction coordinate. Nevertheless, their energetic contribution to the activation free energy represents less than 0.50 kcal/mol for each of the four tested temperatures. Calculation of the transmission coefficient for the isotopically labeled hydride transfer has rendered almost the same values, in agreement with the experimentally observed temperature-independent KIEs. Fourier transform of the time-dependent friction kernel at these four temperatures has allowed obtaining the transition-state friction spectra, which present very small dependence with temperature. Their analysis has led to the identification of some key vibrational modes governing the coupling between the reaction coordinate and the protein environment, thus identifying the relevant motions in the active site and obtaining a full picture of the role of each amino acid.

  6. Orange protein has a role in phytoene synthase stabilization in sweetpotato

    PubMed Central

    Park, Seyeon; Kim, Ho Soo; Jung, Young Jun; Kim, Sun Ha; Ji, Chang Yoon; Wang, Zhi; Jeong, Jae Cheol; Lee, Haeng-Soon; Lee, Sang Yeol; Kwak, Sang-Soo

    2016-01-01

    Carotenoids have essential roles in light-harvesting processes and protecting the photosynthetic machinery from photo-oxidative damage. Phytoene synthase (PSY) and Orange (Or) are key plant proteins for carotenoid biosynthesis and accumulation. We previously isolated the sweetpotato (Ipomoea batatas) Or gene (IbOr), which is involved in carotenoid accumulation and salt stress tolerance. The molecular mechanism underlying IbOr regulation of carotenoid accumulation was unknown. Here, we show that IbOr has an essential role in regulating IbPSY stability via its holdase chaperone activity both in vitro and in vivo. This protection results in carotenoid accumulation and abiotic stress tolerance. IbOr transcript levels increase in sweetpotato stem, root, and calli after exposure to heat stress. IbOr is localized in the nucleus and chloroplasts, but interacts with IbPSY only in chloroplasts. After exposure to heat stress, IbOr predominantly localizes in chloroplasts. IbOr overexpression in transgenic sweetpotato and Arabidopsis conferred enhanced tolerance to heat and oxidative stress. These results indicate that IbOr holdase chaperone activity protects IbPSY stability, which leads to carotenoid accumulation, and confers enhanced heat and oxidative stress tolerance in plants. This study provides evidence that IbOr functions as a molecular chaperone, and suggests a novel mechanism regulating carotenoid accumulation and stress tolerance in plants. PMID:27633588

  7. Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase.

    PubMed

    Abu-Soud, H M; Wang, J; Rousseau, D L; Stuehr, D J

    1999-09-21

    The kinetics of binding L-arginine and three alternative substrates (homoarginine, N-methylarginine, and N-hydroxyarginine) to neuronal nitric oxide synthase (nNOS) were characterized by conventional and stopped-flow spectroscopy. Because binding these substrates has only a small effect on the light absorbance spectrum of tetrahydrobiopterin-saturated nNOS, their binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Im-nNOS with increasing amounts of substrates were obtained and found to be monophasic in all cases. For each substrate, a plot of the apparent rate versus substrate concentration showed saturation at the higher concentrations. K(-)(1), k(2), k(-)(2), and the apparent dissociation constant were derived for each substrate from the kinetic data. The dissociation constants mostly agreed with those calculated from equilibrium spectral data obtained by titrating Im-nNOS with each substrate. We conclude that nNOS follows a two-step, reversible mechanism of substrate binding in which there is a rapid equilibrium between Im-nNOS and the substrate S followed by a slower isomerization process to generate nNOS'-S: Im-nNOS + S if Im-nNOS-S if nNOS'-S + Im. All four substrates followed this general mechanism, but differences in their kinetic values were significant and may contribute to their varying capacities to support NO synthesis.

  8. Stress-induced ER to Golgi translocation of ceramide synthase 1 is dependent on proteasomal processing

    PubMed Central

    Sridevi, Priya; Alexander, Hannah; Laviad, Elad L.; Min, Junxia; Mesika, Adi; Hannink, Mark; Futerman, Anthony H.; Alexander, Stephen

    2009-01-01

    The ceramide synthase (CerS) enzymes are key regulators of ceramide homeostasis. CerS1 is central to regulating C18 ceramide which has been shown to be important in cancer and the response to chemotherapeutic drugs. Previous work indicated that some drugs induced a novel and specific translocation of CerS1 from the endoplasmic reticulum to the Golgi apparatus. We now show that diverse stresses such as UV light, DTT, as well as drugs with different mechanisms of action induce CerS1 translocation. The stresses cause a specific cleavage of the CerS1 enzyme, and the cleavage is dependent on the action of the proteasome. Inhibition of proteasome function inhibits stress-induced CerS1 translocation, indicating that this proteolytic cleavage precedes the translocation. Modulation of protein kinase C activity shows that it plays a central role in regulating CerS1 translocation. Analysis of the C-terminus of the CerS1 protein shows that several KxKxx motifs are not involved in regulating stress induced translocation. The study suggests that diverse stresses initiate responses through different signaling pathways, which ultimately converge to regulate CerS1 localization. The data provide an increasingly detailed understanding of the regulation of this important enzyme in normal and stressed cells, and support the idea that it is uniquely regulated with respect to the other CerS enzymes. PMID:19800881

  9. Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

    PubMed

    Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

    2017-03-01

    Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Indazole, Pyrazole, and Oxazole Derivatives Targeting Nitric Oxide Synthases and Carbonic Anhydrases.

    PubMed

    Maccallini, Cristina; Di Matteo, Mauro; Vullo, Daniela; Ammazzalorso, Alessandra; Carradori, Simone; De Filippis, Barbara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Pandolfi, Assunta; Supuran, Claudiu T; Amoroso, Rosa

    2016-08-19

    Nitric oxide (NO) is an essential endogenous mediator with a physiological role in the central nervous system as neurotransmitter and neuromodulator. A growing number of studies have demonstrated that abnormal nitrergic signaling is a crucial event in the development of neurodegeneration. In particular, the uncontrolled production of NO by neuronal nitric oxide synthase (nNOS) is observed in several neurodegenerative diseases. Moreover, it is well recognized that specific isoforms of human carbonic anhydrase (hCA) physiologically modulate crucial pathways of signal processing and that low expression of CA affects cognition, leading to mental retardation, Alzheimer's disease, and aging-related cognitive impairments. In light of this, dual agents that are able to target both NOS (inhibition) and CA (activation) could be useful drug candidates for the treatment of Alzheimer's disease, aging, and other neurodegenerative diseases. In the present work, we show the design, synthesis, and in vitro biological evaluation of new nitrogen-based heterocyclic compounds. Among the tested molecules, 2-amino-3-(4-hydroxyphenyl)-N-(1H-indazol-5-yl)propanamide hydrochloride (10 b) was revealed to be a potent dual agent, able to act as a selective nNOS inhibitor and activator of the hCA I isoform. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Inhibition of chalcone synthase Expression in Anthers of Raphanus sativus with Ogura Male Sterile Cytoplasm

    PubMed Central

    Yang, Soojung; Terachi, Toru; Yamagishi, Hiroshi

    2008-01-01

    Background and Aims Expression of the mitochondrial gene orf138 causes Ogura cytoplasmic male sterility (CMS) in Raphanus sativus, but little is known about the mechanism by which CMS takes place. A preliminary microarray experiment revealed that several nuclear genes concerned with flavonoid biosynthesis were inhibited in the male-sterile phenotype. In particular, a gene for one of the key enzymes for flavonoid biosynthesis, chalcone synthase (CHS), was strongly inhibited. A few reports have suggested that the inhibition of CHS causes nuclear-dependent male sterile expression; however, there do not appear to be any reports elucidating the effect of CHS on CMS expression. In this study, the expression patterns of the early genes in the flavonoid biosynthesis pathway, including CHS, were investigated in normal and male-sterile lines. Methods In order to determine the aberrant stage for CMS expression, the characteristics of male-sterile anthers are observed using light and transmission electron microscopy for several stages of flower buds. The expression of CHS and the other flavonoid biosynthetic genes in the anthers were compared between normal and male-sterile types using real time RT-PCR. Key Results Among the flavonoid biosynthetic genes analysed, the expression of CHS was strongly inhibited in the later stages of anther development in sterility cytoplasm; accumulation of putative naringenin derivatives was also inhibited. Conclusions These results show that flavonoids play an important role in the development of functional pollen, not only in nuclear-dependent male sterility, but also in CMS. PMID:18625698

  12. A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

    PubMed Central

    Tidhar, Rotem; Sims, Kacee; Rosenfeld-Gur, Eden; Shaw, Walter; Futerman, Anthony H.

    2015-01-01

    Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases. PMID:25368106

  13. Comparative structural and computational analysis supports eighteen cellulose synthases in the plant cellulose synthesis complex

    SciTech Connect

    Nixon, B. Tracy; Mansouri, Katayoun; Singh, Abhishek; Du, Juan; Davis, Jonathan K.; Lee, Jung -Goo; Slabaugh, Erin; Vandavasi, Venu Gopal; O’Neill, Hugh Michael; Roberts, Eric M.; Roberts, Alison W.; Yingling, Yaroslava G.; Haigler, Candace H.

    2016-06-27

    A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In conclusion, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.

  14. Measuring Light

    NASA Image and Video Library

    2017-09-27

    On July 13, 2011, Don Perovich, of Cold Regions Research and Engineering Laboratory, measured the light that drives photosynthesis at the sixth sea ice station of the 2011 ICESCAPE mission. The ICESCAPE mission, or "Impacts of Climate on Ecosystems and Chemistry of the Arctic Pacific Environment," is a NASA shipborne investigation to study how changing conditions in the Arctic affect the ocean's chemistry and ecosystems. The bulk of the research took place in the Beaufort and Chukchi seas in summer 2010 and 2011. Credit: NASA/Kathryn Hansen NASA image use policy. NASA Goddard Space Flight Center enables NASA’s mission through four scientific endeavors: Earth Science, Heliophysics, Solar System Exploration, and Astrophysics. Goddard plays a leading role in NASA’s accomplishments by contributing compelling scientific knowledge to advance the Agency’s mission. Follow us on Twitter Like us on Facebook Find us on Instagram

  15. Light Echo

    NASA Image and Video Library

    2017-09-27

    "Light Echo" Illuminates Dust Around Supergiant Star V838 Monocerotis (V838 Mon) Credit: NASA and The Hubble Heritage Team (AURA/STScI) The Hubble Space Telescope is a project of international cooperation between NASA and the European Space Agency. NASA's Goddard Space Flight Center manages the telescope. The Space Telescope Science Institute conducts Hubble science operations. Goddard is responsible for HST project management, including mission and science operations, servicing missions, and all associated development activities. To learn more about the Hubble Space Telescope go here: www.nasa.gov/mission_pages/hubble/main/index.html NASA Goddard Space Flight Center is home to the nation's largest organization of combined scientists, engineers and technologists that build spacecraft, instruments and new technology to study the Earth, the sun, our solar system, and the universe. Follow us on Twitter Join us on Facebook

  16. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.

    PubMed

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-06-01

    Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

  17. Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase.

    PubMed

    Cushman, Mark; Yang, Donglai; Gerhardt, Stefan; Huber, Robert; Fischer, Markus; Kis, Klaus; Bacher, Adelbert

    2002-08-09

    A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase. The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3'-carboxypropyl compound 22. Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity. In the phosphonoxyalkyl series, the 3'-phosphonoxypropyl compound 33 was more potent than the 4'-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3'-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2'-carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.

  18. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    PubMed

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  19. Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum.

    PubMed

    Li, Pan-Pan; Li, De-Feng; Liu, Di; Liu, Yi-Ming; Liu, Chang; Liu, Shuang-Jiang

    2013-12-01

    Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg.

  20. Functional contribution of chorismate synthase, anthranilate synthase, and chorismate mutase to penetration resistance in barley-powdery mildew interactions.

    PubMed

    Hu, Pingsha; Meng, Yan; Wise, Roger P

    2009-03-01

    Plant processes resulting from primary or secondary metabolism have been hypothesized to contribute to defense against microbial attack. Barley chorismate synthase (HvCS), anthranilate synthase alpha subunit 2 (HvASa2), and chorismate mutase 1 (HvCM1) occupy pivotal branch points downstream of the shikimate pathway leading to the synthesis of aromatic amino acids. Here, we provide functional evidence that these genes contribute to penetration resistance to Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. Single-cell transient-induced gene silencing of HvCS and HvCM1 in mildew resistance locus a (Mla) compromised cells resulted in increased susceptibility. Correspondingly, overexpression of HvCS, HvASa2, and HvCM1 in lines carrying mildew resistance locus o (Mlo), a negative regulator of penetration resistance, significantly decreased susceptibility. Barley stripe mosaic virus-induced gene silencing of HvCS, HvASa2, and HvCM1 significantly increased B. graminis f. sp. hordei penetration into epidermal cells, followed by formation of haustoria and secondary hyphae. However, sporulation of B. graminis f. sp. hordei was not detected on the silenced host plants up to 3 weeks after inoculation. Taken together, these results establish a previously unrecognized role for the influence of HvCS, HvASa2, and HvCM1 on penetration resistance and on the rate of B. graminis f. sp. hordei development in Mla-mediated, barley-powdery mildew interactions.

  1. Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

    PubMed

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

    2012-12-05

    Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

  2. Functional Specificity of Cardiolipin Synthase Revealed by the Identification of a Cardiolipin Synthase CrCLS1 in Chlamydomonas reinhardtii

    PubMed Central

    Hung, Chun-Hsien; Kobayashi, Koichi; Wada, Hajime; Nakamura, Yuki

    2016-01-01

    Phosphatidylglycerol (PG) and cardiolipin (CL) are two essential classes of phospholipid in plants and algae. Phosphatidylglycerophosphate synthase (PGPS) and cardiolipin synthase (CLS) involved in the biosynthesis of PG and CL belong to CDP-alcohol phosphotransferase and share overall amino acid sequence homology. However, it remains elusive whether PGPS and CLS are functionally distinct in vivo. Here, we report identification of a gene encoding CLS in Chlamydomonas reinhardtii, CrCLS1, and its functional compatibility. Whereas CrCLS1 did not complement the growth phenotype of a PGPS mutant of Synechocystis sp. PCC 6803, it rescued the temperature-sensitive growth phenotype, growth profile with different carbon sources, phospholipid composition and enzyme activity of Δcrd1, a CLS mutant of Saccharomyces cerevisiae. These results suggest that CrCLS1 encodes a functional CLS of C. reinhardtii as the first identified algal CLS, whose enzyme function is distinct from that of PGPSs from C. reinhardtii. Comparison of CDP-alcohol phosphotransferase motif between PGPS and CLS among different species revealed a possible additional motif that might define the substrate specificity of these closely related enzymes. PMID:26793177

  3. Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes.

    PubMed

    Nurhayati, Niknik; Gondé, Daniela; Ober, Dietrich

    2009-03-01

    In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant's constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitment occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a "processed pseudogene", and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.

  4. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

    PubMed

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-03-14

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.

  5. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway

    PubMed Central

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. PMID:26971881

  6. A gene from the cellulose synthase-like C family encodes a β-1,4 glucan synthase

    PubMed Central

    Cocuron, Jean-Christophe; Lerouxel, Olivier; Drakakaki, Georgia; Alonso, Ana P.; Liepman, Aaron H.; Keegstra, Kenneth; Raikhel, Natasha; Wilkerson, Curtis G.

    2007-01-01

    Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the β-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a β-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone. PMID:17488821

  7. Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

    NASA Astrophysics Data System (ADS)

    Xie, Qiao-Wen; Cho, Hearn J.; Calaycay, Jimmy; Mumford, Richard A.; Swiderek, Kristine M.; Lee, Terry D.; Ding, Aihao; Troso, Tiffany; Nathan, Carl

    1992-04-01

    Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

  8. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

    PubMed

    Maruyama, T; Ito, M; Honda, G

    2001-10-01

    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  9. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    PubMed

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata.

  10. Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression.

    PubMed

    Lu, Shan; Xu, Ran; Jia, Jun-Wei; Pang, Jihai; Matsuda, Seiichi P T; Chen, Xiao-Ya

    2002-09-01

    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 (-)-beta-pinene and (-)-alpha-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 (-)-beta-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.

  11. Disruption of glycogen synthase kinase-3-beta activity leads to abnormalities in physiological measures in mice.

    PubMed

    Ahnaou, A; Drinkenburg, W H I M

    2011-08-01

    Dysregulation of glycogen synthase kinase-3-beta (GSK-3β) signaling pathways is thought to underlie the pathophysiology of mood disorders. In order to demonstrate that the loss of normal GSK-3β activity results in disturbances of physiological measures, we attempted to determine whether sleep-wake architecture, circadian rhythms of core body temperature and activity were altered in transgenic mice overexpressing GSK-3β activity specifically in the brain. Cortical electroencephalographic activity, body temperature (BT) and body locomotor activity (LMA) were continuously monitored using a biopotential telemetry probe. Normal circadian patterns were maintained for different measurements in both genotypes. No differences were found in total time spent asleep and waking over the 24-h recording session. However, transgenic animals overexpressing GSK-3β showed alteration in sleep continuity characterized by an increases in number of non rapid eye movement (NREM) sleep episodes (GSK-3β, 227.2 ± 1.7 vs. WT, 172.6 ± 1.4, p < 0.05) and decreases in mean episode duration (GSK-3β, 3.0 ± 0.1 vs. WT, 4.4 ± 0.2, p < 0.05). Additionally, transgenic animals exhibited marked enhancement of basal LMA and BT levels during the first part of the dark period, under both light-dark and free running dark-dark circadian cycles. Our findings indicate that transgenic mice overexpressing GSK-3β activity exhibit severe fragmentation of sleep-wake cycle during both the light and dark periods, without showing deviancy in total durations of vigilance states. The results strongly suggest that GSK-3β activity is elemental for the maintenance of circadian motor behavior levels required for proper regulation of BT and sleep-wake organization.

  12. Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

    PubMed

    Smith, Amber Marie; Brown, William Clay; Harms, Etti; Smith, Janet L

    2015-02-27

    PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.

  13. Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides.

    PubMed

    Radcliffe, C W; Steiner, F X; Carman, G M; Niederman, R A

    1989-01-01

    Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent Km values of PGP synthase were 90 microM for sn-glycerol-3-phosphate and 60 microM for CDP-diacylglycerol; the apparent Km of PS synthase for L-serine was near 165 microM. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10-50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase was distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromato-focusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C.

  14. (E)-β-Ocimene and Myrcene Synthase Genes of Floral Scent Biosynthesis in Snapdragon

    PubMed Central

    Dudareva, Natalia; Martin, Diane; Kish, Christine M.; Kolosova, Natalia; Gorenstein, Nina; Fäldt, Jenny; Miller, Barbara; Bohlmann, Jörg

    2003-01-01

    Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-β-ocimene, derived from geranyl diphosphate, in ad-dition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-β-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-β-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-β-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-β-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx8W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production. PMID:12724546

  15. Lighting the Learning Environment.

    ERIC Educational Resources Information Center

    Fielding, Randall

    2000-01-01

    Explores the benefits and pitfalls of day lighting, indirect light, and full-spectrum lamps for general illumination and accent lighting in classrooms. Discussions include lighting considerations in areas where computers are used and fixture cost factors versus efficiency. (GR)

  16. Lighting the Learning Environment.

    ERIC Educational Resources Information Center

    Fielding, Randall

    2000-01-01

    Explores the benefits and pitfalls of day lighting, indirect light, and full-spectrum lamps for general illumination and accent lighting in classrooms. Discussions include lighting considerations in areas where computers are used and fixture cost factors versus efficiency. (GR)

  17. Traffic Lights: Red Light Spells Danger.

    ERIC Educational Resources Information Center

    Reed, Chris

    2001-01-01

    A "traffic light" model provides youth groups with a means to evaluate the risk level of specific behaviors and agree upon the management of such behaviors. Designed for outdoor pursuits, the model may be used in other environments. Suggestions for ways to discuss red-light, yellow-light, and green-light behaviors are included. (SV)

  18. Depletion of stromal P(i) induces high 'energy-dependent' antenna exciton quenching (q(E)) by decreasing proton conductivity at CF(O)-CF(1) ATP synthase.

    PubMed

    Takizawa, Kenji; Kanazawa, Atsuko; Kramer, David M

    2008-02-01

    This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P(i)), which results in down-regulation of light capture via the exciton quenching (q(E)) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P(i) depletion. In one model, an imbalance of ATP or NADPH activa