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Sample records for 5-phosphate synthase dxs

  1. Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans.

    PubMed

    Henriquez, Maria Antonia; Soliman, Atta; Li, Genyi; Hannoufa, Abdelali; Ayele, Belay T; Daayf, Fouad

    2016-02-01

    1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the plastidial 2C-methyl-D-erythritol-4-phosphate (DOXP-MEP) pathway involved in isoprenoid biosynthesis. In this study, we cloned the complete cDNA of potato DXS gene that was designated StDXS1. StDXS1 cDNA encodes for 719 amino acid residues, with MW of 77.8 kDa, and is present in one copy in the potato genome. Phylogenetic analysis and protein sequence alignments assigned StDXS1 to a group with DXS homologues from closely related species and exhibited homodomain identity with known DXS proteins from other plant species. Late blight symptoms occurred in parallel with a reduction in StDXS1 transcript levels, which may be associated with the levels of isoprenoids that contribute to plant protection against pathogens. Subcellular localization indicated that StDXS1 targets the chloroplasts where isoprenoids are synthesized. Arabidopsis expressing StDXS1 showed a higher accumulation of carotenoids and chlorophyll as compared to wild type controls. Lower levels of ABA and GA were detected in the transgenic DXS lines as compared to control plants, which reflected on higher germination rates of the transgenic DXS lines. No changes were detected in JA or SA contents. Selected downstream genes in the DOXP-MEP pathway, especially GGPPS genes, were up-regulated in the transgenic lines.

  2. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    PubMed Central

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  3. The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation.

    PubMed

    Peng, Gang; Wang, Chunyan; Song, Song; Fu, Xiumin; Azam, Muhammad; Grierson, Don; Xu, Changjie

    2013-10-01

    Three 1-deoxy-D-xylulose-5-phosphate synthases (DXS) and three phytoene synthases (PSY) were identified in citrus, from Affymetrix GeneChip Citrus Genome Array, GenBank and public orange genome databases. Tissue-specific expression analysis of these genes was carried out on fruit peel and flesh, flower and leaf of Satsuma mandarin (Citrus unshiu Marc.) in order to determine their roles in carotenoid accumulation in different tissues. Expression of CitDXS1 and CitPSY1 was highest in all test tissues, while that of CitDXS2 and CitPSY2 was lower, and that of CitDXS3 and CitPSY3 undetectable. The transcript profiles of CitDXS1 and CitPSY1 paralleled carotenoid accumulation in flesh of Satsuma mandarin and orange (Citrus sinensis Osbeck) during fruit development, and CitPSY1 expression was also associated with carotenoid accumulation in peel, while the CitDXS1 transcript level was only weakly correlated with carotenoid accumulation in peel. Similar results were obtained following correlation analysis between expression of CitDXS1 and CitPSY1 and carotenoid accumulation in peel and flesh of 16 citrus cultivars. These findings identify CitPSY1 and CitDXS1 as the main gene members controlling carotenoid biosynthesis in citrus fruit. Furthermore, chromoplasts were extracted from flesh tissue of these citrus, and chromoplasts of different shape (spindle or globular), different size, and color depth were observed in different cultivars, indicating chromoplast abundance, number per gram tissue, size and color depth were closely correlated with carotenoid content in most cultivars. The relationship between carotenoid biosynthesis and chromoplast development was discussed.

  4. Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.

    PubMed

    Banerjee, Aparajita; Wu, Yan; Banerjee, Rahul; Li, Yue; Yan, Honggao; Sharkey, Thomas D

    2013-06-01

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

  5. Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis1[C][W][OPEN

    PubMed Central

    Wright, Louwrance P.; Rohwer, Johann M.; Ghirardo, Andrea; Hammerbacher, Almuth; Ortiz-Alcaide, Miriam; Raguschke, Bettina; Schnitzler, Jörg-Peter; Gershenzon, Jonathan; Phillips, Michael A.

    2014-01-01

    The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway. PMID:24987018

  6. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize.

    PubMed

    Cordoba, Elizabeth; Porta, Helena; Arroyo, Analilia; San Román, Carolina; Medina, Luis; Rodríguez-Concepción, Manuel; León, Patricia

    2011-03-01

    The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme catalyses the first biosynthetic step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In plants the MEP pathway is involved in the synthesis of the common precursors to the plastidic isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate, in plastids. DXS is recognized as limiting this pathway and is a potential target for manipulation to increase various isoprenoids such as carotenoids. In Zea mays three dxs genes exist that encode plastid-targeted functional enzymes. Evidence is provided that these genes represent phylogenetically distinctive clades conserved among plants preceding monocot-dicot divergence. There is differential accumulation for each dxs gene transcript, during development and in response to external signals such as light. At the protein level, the analysis demonstrates that in Z. mays, DXS protein is feedback regulated in response to the inhibition of the pathway flow. The results support that the multilevel regulation of DXS activity is conserved in evolution.

  7. Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana.

    PubMed

    Carretero-Paulet, Lorenzo; Cairó, Albert; Talavera, David; Saura, Andreu; Imperial, Santiago; Rodríguez-Concepción, Manuel; Campos, Narciso; Boronat, Albert

    2013-07-15

    The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.

  8. Molecular cloning and characterization of three cDNAs encoding 1-deoxy-d-xylulose-5-phosphate synthase in Aquilaria sinensis (Lour.) Gilg.

    PubMed

    Xu, Yanhong; Liu, Juan; Liang, Liang; Yang, Xin; Zhang, Zheng; Gao, Zhihui; Sui, Chun; Wei, Jianhe

    2014-09-01

    Agarwood is an expensive resinous heartwood derived from Aquilaria plants that is widely used in traditional medicines, incense and perfume. The major constituents of agarwood oils are sesquiterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-deoxy-d-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme for sesquiterpene synthesis in the MEP pathway. In this study, 3 cDNAs of DXS genes were cloned and characterized from the Aquilaria sinensis (Lour.) Gilg. These genes represent 3 phylogenetically distinct clades conserved among plants. Functional complementation in a DXS-deficient Escherichia coli strain EcAB4-2 demonstrated that they are active DXS, which rescued the E. coli mutant. Their expression profiles in different tissues and in response to different treatments were analyzed by real-time PCR. All 3 genes are highly expressed in stem, followed by leaf and root. AsDXS1 was significantly stimulated by mechanical, chemical, and H2O2 treatment, whereas AsDXS2 and AsDXS3 only responded to chemical treatment and mechanical treatment, respectively. All three genes were oscillation in respond to MJ treatment, with expression peaks occurring at different time points. Our results suggest the conservation of DXS in evolution and imply their distinct functions in primary and defensive sesquiterpene metabolism in A. sinensis.

  9. Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis

    SciTech Connect

    Xiang,S.; Usunow, G.; Busch, G.; Tong, L.

    2007-01-01

    Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.

  10. Thiamin Diphosphate Activation in 1-Deoxy-d-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions.

    PubMed

    White, Justin K; Handa, Sumit; Vankayala, Sai Lakshmana; Merkler, David J; Woodcock, H Lee

    2016-09-22

    1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4'-aminopyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4'-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ∼10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.

  11. Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.

    PubMed

    Kudoh, Kai; Kawano, Yusuke; Hotta, Shingo; Sekine, Midori; Watanabe, Takafumi; Ihara, Masaki

    2014-07-01

    Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.

  12. The isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 controls isoprenoid profiles, precursor pathway allocation, and density of tomato trichomes.

    PubMed

    Paetzold, Heike; Garms, Stefan; Bartram, Stefan; Wieczorek, Jenny; Urós-Gracia, Eva-Maria; Rodríguez-Concepción, Manuel; Boland, Wilhelm; Strack, Dieter; Hause, Bettina; Walter, Michael H

    2010-09-01

    Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SlDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SlDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SlDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SlDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ¹³C to ¹²C natural isotope ratios. A compensatory up-regulation of SlDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation. PMID:20591838

  13. Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle.

    PubMed

    Morris, Wayne L; Ducreux, Laurence J M; Hedden, Peter; Millam, Steve; Taylor, Mark A

    2006-01-01

    Potato tubers were engineered to express a bacterial gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in order to investigate the effects of perturbation of isoprenoid biosynthesis. Twenty-four independent transgenic lines out of 38 generated produced tubers with significantly elongated shape that also exhibited an early tuber sprouting phenotype. Expression analysis of nine transgenic lines (four exhibiting the phenotype and five showing a wild-type phenotype) demonstrated that the phenotype was strongly associated with dxs expression. At harvest, apical bud growth had already commenced in dxs-expressing tubers whereas in control lines no bud growth was evident until dormancy was released after 56-70 d of storage. The initial phase of bud growth in dxs tubers was followed by a lag period of approximately 56 d, before further elongation of the developing sprouts could be detected. Thus dxs expression results in the separation of distinct phases in the dormancy and sprouting processes. In order to account for the sprouting phenotype, the levels of plastid-derived isoprenoid growth regulators were measured in transgenic and control tubers. The major difference measured was an increase in the level of trans-zeatin riboside in tubers at harvest expressing dxs. Additionally, compared with controls, in some dxs-expressing lines, tuber carotenoid content increased approximately 2-fold, with most of the increase accounted for by a 6-7-fold increase in phytoene. PMID:16873449

  14. Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii

    PubMed Central

    Tong, Yuru; Su, Ping; Zhao, Yujun; Zhang, Meng; Wang, Xiujuan; Liu, Yujia; Zhang, Xianan; Gao, Wei; Huang, Luqi

    2015-01-01

    1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.KM879187), 2148 bp (TwDXS2, GenBank accession no.KM879186), 1410 bp (TwDXR, GenBank accession no.KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g−1, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f. PMID:26512659

  15. Mechanistic binding insights for 1-deoxy-D-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Battistini, Matthew R; Shoji, Christopher; Handa, Sumit; Breydo, Leonid; Merkler, David J

    2016-04-01

    We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

  16. Helper component-proteinase enhances the activity of 1-deoxy-D-xylulose-5-phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y-infected tobacco.

    PubMed

    Li, Heng; Ma, Dongyuan; Jin, Yongsheng; Tu, Yayi; Liu, Liping; Leng, Chunxu; Dong, Jiangli; Wang, Tao

    2015-10-01

    Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships.

  17. 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants.

    PubMed

    Estévez, J M; Cantero, A; Reindl, A; Reichler, S; León, P

    2001-06-22

    The initial step of the plastidic 2C-methyl-D-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-D-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production.

  18. Hydroxybenzaldoximes are d-GAP-competitive inhibitors of E. coli 1-deoxy-d-xylulose-5-phosphate synthase

    PubMed Central

    Bartee, David; Morris, Francine; Al-khouja, Amer

    2015-01-01

    1-Deoxy-d-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branchpoint, serving also as a precursor in the biosynthesis of vitamins B1 and B6 which are critical for central metabolism. Toward identifying novel bisubstrate analog inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low micromolar inhibitors, competitive against d-glyceraldehyde 3-phosphate (d-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of d-GAP-competitive DXP synthase inhibitors offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis. PMID:26174207

  19. Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

    PubMed

    Bartee, David; Morris, Francine; Al-Khouja, Amer; Freel Meyers, Caren L

    2015-08-17

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.

  20. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine.

    PubMed

    Choudhury, Swarup Roy; Singh, Sanjay Kumar; Roy, Sujit; Sengupta, Dibyendu N

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP. PMID:20689184

  1. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine.

    PubMed

    Choudhury, Swarup Roy; Singh, Sanjay Kumar; Roy, Sujit; Sengupta, Dibyendu N

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP.

  2. Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Jawaid, Safdar; Seidle, Heather; Zhou, Weidong; Abdirahman, Hafsa; Abadeer, Maher; Hix, Joseph H.; van Hoek, Monique L.; Couch, Robin D.

    2009-01-01

    Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis. PMID:20011597

  3. Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine β-synthase.

    PubMed

    Yadav, Pramod Kumar; Xie, Peter; Banerjee, Ruma

    2012-11-01

    Human cystathionine β-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

  4. Population data for DXS6800, DXS101 and DXS8377 loci from Buenos Aires (Argentina).

    PubMed

    Noseda, Pablo A; Kenline, Jaclyn; Manning, Samantha; Gangitano, David A

    2013-07-01

    The X-chromosomal short tandem repeats (X-STRs) DXS6800, DXS101 and DXS8377 were analysed in a population sample from Buenos Aires (Argentina) using a polymerase chain reaction (PCR) multiplex approach with fluorescent detection. We present allele frequencies for these loci in a population comprising 113 women and 99 men. The Hardy-Weinberg equilibrium (HWE) was tested in the female sample and no significant deviations were observed. The homogeneity of allele frequencies of men and women was compared by the Fisher's exact test and showed similar distributions. Linkage disequilibrium (LD) tests were performed in males for all pairs of loci and no significant associations were detected. Parameters of forensic interest were also estimated.

  5. Population data for DXS6800, DXS101 and DXS8377 loci from Buenos Aires (Argentina).

    PubMed

    Noseda, Pablo A; Kenline, Jaclyn; Manning, Samantha; Gangitano, David A

    2013-07-01

    The X-chromosomal short tandem repeats (X-STRs) DXS6800, DXS101 and DXS8377 were analysed in a population sample from Buenos Aires (Argentina) using a polymerase chain reaction (PCR) multiplex approach with fluorescent detection. We present allele frequencies for these loci in a population comprising 113 women and 99 men. The Hardy-Weinberg equilibrium (HWE) was tested in the female sample and no significant deviations were observed. The homogeneity of allele frequencies of men and women was compared by the Fisher's exact test and showed similar distributions. Linkage disequilibrium (LD) tests were performed in males for all pairs of loci and no significant associations were detected. Parameters of forensic interest were also estimated. PMID:23756526

  6. Defining critical residues for substrate binding to 1-deoxy-D-xylulose 5-phosphate synthase--active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate.

    PubMed

    Brammer Basta, Leighanne A; Patel, Hetalben; Kakalis, Lazaros; Jordan, Frank; Freel Meyers, Caren L

    2014-06-01

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GraP) in a thiamin diphosphate-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study used mutagenesis and a combination of protein fluorescence, CD and kinetics experiments to investigate the roles of Arg420, Arg478 and Tyr392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism, and predict that Arg420 and Arg478 are involved in binding of the acceptor substrate, GraP. D-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg420 and Arg478, also accelerates decarboxylation of the predecarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating that this binding interaction is not absolutely required, and that the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr392 contributes to GraP affinity, and is not required for LThDP formation or its GraP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate that LThDP is significantly stabilized on R420A and Y392F variants as compared with wild-type DXP synthase in the absence of acceptor substrate, but these substitutions do not appear to affect the rate of GraP-promoted LThDP decarboxylation in the presence of high levels of GraP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues in promoting GraP binding, which in turn facilitates decarboxylation, and also highlight interesting differences between DXP synthase and other thiamin diphosphate-dependent enzymes.

  7. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme. PMID:25151124

  8. Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy-D-xylulose 5-phosphate synthase: 600-fold rate acceleration of pyruvate decarboxylation by D-glyceraldehyde-3-phosphate

    PubMed Central

    Patel, Hetalben; Nemeria, Natalia S.; Brammer, Leighanne A.; Freel Meyers, Caren L.; Jordan, Frank

    2012-01-01

    The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as 2-hydroxyethyl donor with D-glyceraldehyde-3-phosphate (D-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and pre-steady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound pre-decarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved CD spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design. PMID:23072514

  9. Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.

    PubMed

    Estévez, J M; Cantero, A; Romero, C; Kawaide, H; Jiménez, L F; Kuzuyama, T; Seto, H; Kamiya, Y; León, P

    2000-09-01

    The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The beta-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.

  10. Ribose-5-phosphate isomerase and ribulose-5-phosphate kinase show apparent specificity for a specific ribulose 5-phosphate species.

    PubMed

    Anderson, L E

    1987-02-01

    Ribose-5-phosphate isomerase and ribulose-5-phosphate kinase appear to show specificity for a particular ribulose 5-phosphate species. The effect of this specificity will be channeling of ribulose 5-phosphate from the isomerase to the kinase during photosynthesis.

  11. The gene for spondyloepiphyseal dysplasia (SEDL) maps to Xp22 between DXS16 and DXS92

    SciTech Connect

    Heuertz, S.; Le Merrer, M.; Delrieu, O. ); Nelen, M.; Hamel, B.; Van Oost, B.A. ); Wilkie, A.O.M. ); Larget-Piet, L. ); Tranebjaerg, L. ); Bick, D. )

    1993-10-01

    Previous linkage studies in X-linked spondyloepiphyseal dysplasia (SEDL) placed the gene in the region Xp22.2-p22.1 by linkage to DXS41. Here, the authors have extended earlier studies by analyzing 15 families with 13 markers from the Xp2 region. Pairwise linkage analysis revealed significant linkage of the SEDL to 8 markers from the Xp22.2-Xp22.1 region. Maximum lod scores were obtained with DXS207, z[sub max] = 9.16 at [theta][sub max] = 0.021 with confidence limits of 0.00-0.09, and DXS197, z[sub max] = 7.98 at [theta][sub max] = 0.00 with confidence limits of 0.00-0.06. The study of one recombinant in family 4 indicated that DXS 41 is more likely proximal to DXS92 than distal. Multipoint linkage results and analysis of recombination events indicated that the mutation responsible for SEDL is located in Xp22 between DXS16 and DXS92. 39 refs., 3 figs., 2 tabs.

  12. Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase.

    PubMed

    Carretero-Paulet, Lorenzo; Cairó, Albert; Botella-Pavía, Patricia; Besumbes, Oscar; Campos, Narciso; Boronat, Albert; Rodríguez-Concepción, Manuel

    2006-11-01

    The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.

  13. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    PubMed

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  14. Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies).

    PubMed

    Zulak, Katherine G; Lippert, Dustin N; Kuzyk, Michael A; Domanski, Dominik; Chou, Tina; Borchers, Christoph H; Bohlmann, Jörg

    2009-12-01

    Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono- and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce.

  15. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  16. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  17. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  18. Genetics Home Reference: pyridoxal 5'-phosphate-dependent epilepsy

    MedlinePlus

    ... Clayton PT, Baumgartner MR, Steinmann B, Bast T, Wolf NI, Zschocke J. Pyridoxal 5'-phosphate may be ... Clayton PT, Baumgartner MR, Steinmann B, Bast T, Wolf NI, Zschocke J. Pyridoxal 5'-phosphate may be ...

  19. Arabinose 5-phosphate covalently inhibits transaldolase.

    PubMed

    Light, Samuel H; Anderson, Wayne F

    2014-03-01

    Arabinose 5-phosphate (A5P) is the aldopentose version of the ketohexose fructose 6-phosphate (F6P), having identical stereochemistry but lacking atoms corresponding to the 1-carbon and 1-hydroxyl. Despite structural similarity and conservation of the reactive portion of F6P, F6P acts as a substrate whereas A5P is reported to be an inhibitor of transaldolase. To address the lack of A5P reactivity we determined a crystal structure of the Francisella tularensis transaldolase in complex with A5P. This structure reveals that like F6P, A5P forms a covalent Schiff base with active site Lys135. Unlike F6P, A5P binding fails to displace an ordered active site water molecule. Retaining this water necessitates conformational changes at the A5P-protein linkage that possibly hinder reactivity. The findings presented here show the basis of A5P inhibition and suggest an unusual mechanism of competitive, reversible-covalent transaldolase regulation.

  20. The measurement of xylulose 5-phosphate, ribulose 5-phosphate, and combined sedoheptulose 7-phosphate and ribose 5-phosphate in liver tissue.

    PubMed

    Casazza, J P; Veech, R L

    1986-12-01

    A modification of the method of Kauffman et al. (F. C. Kauffman, J. G. Brown, J. V. Passonneau, and O. H. Lowry (1969) J. Biol. Chem. 244, 3647-3653) for the spectrophotometric determination of xylulose 5-phosphate, ribulose 5-phosphate, and combined ribose 5-phosphate and sedoheptulose 7-phosphate in tissue extract is presented. Using commercially available enzymes all three assays come to a clear endpoint with the assays described. Values for these metabolites in liver in three dietary states are reported; 48 h starved, ad libitum feeding of standard NIH rat ration, and meal feeding of a fat-free diet. Xylulose 5-phosphate values were 3.8 +/- 0.3, 8.6 +/- 0.3, and 66.3 +/- 8.3 nmol/g. Ribulose 5-phosphate values were 3.4 +/- 0.3, 5.8 +/- 0.2, and 37.1 +/- 5.3 nmol/g. Combined ribose 5-phosphate and sedoheptulose 7-phosphate were 29.3 +/- 0.3, 38.2 +/- 1.2, and 108.2 +/- 14.5 nmol/g. The ratio of measured tissue content of [xylulose 5-phosphate]/[ribulose 5-phosphate] was found to be 1.12 +/- 0.07 in starved animals, 1.48 +/- 0.04 in ad libitum fed animals and 1.78 +/- 0.03 in low-fat meal fed animals. These data are in good agreement with the range of equilibrium constants reported for this reaction, suggesting that the ribulose 5-phosphate 3-epimerase reaction (EC 5.1.3.1) is a near equilibrium reaction despite a more than 10-fold change in the tissue content of these metabolites.

  1. Pyridoxal-5'-phosphate-dependent catalytic antibodies.

    PubMed

    Gramatikova, Svetlana; Mouratou, Barbara; Stetefeld, Jörg; Mehta, Perdeep K; Christen, Philipp

    2002-11-01

    Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5'-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N(alpha)-(5'-phosphopyridoxyl)-L-lysine, a stable analog of the covalent coenzyme-substrate adducts were screened by a competition ELISA for binding of the PLP-amino acid Schiff base adduct. The Schiff base with its C4'-N alpha double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent alpha,beta-elimination reaction of beta-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k(cat)'=0.42 min(-1) with D-alanine at 25 degrees C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme-amino acid adduct. (2) The bond at C alpha to be broken lies together with the C alpha-N bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The alpha-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme-substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10(4) times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k(cat (Ab x PLP))'/k(cat (PLP))'=5 x 10(3)). The analogies of antibody 15A9 with

  2. Natural Variation in Monoterpene Synthesis in Kiwifruit: Transcriptional Regulation of Terpene Synthases by NAC and ETHYLENE-INSENSITIVE3-Like Transcription Factors1

    PubMed Central

    Nieuwenhuizen, Niels J.; Chen, Xiuyin; Wang, Mindy Y.; Matich, Adam J.; Perez, Ramon Lopez; Allan, Andrew C.; Green, Sol A.; Atkinson, Ross G.

    2015-01-01

    Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-d-erythritol 4-phosphate pathway enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-d-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits. PMID:25649633

  3. Natural variation in monoterpene synthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.

    PubMed

    Nieuwenhuizen, Niels J; Chen, Xiuyin; Wang, Mindy Y; Matich, Adam J; Perez, Ramon Lopez; Allan, Andrew C; Green, Sol A; Atkinson, Ross G

    2015-04-01

    Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.

  4. No evidence for linkage between the X-chromosome marker DXS7 and schizophrenia

    SciTech Connect

    Okoro, C.; Bell, R.; Sham, P.; Asherson, P.

    1995-10-09

    DeLisi et al. have examined the X and Y chromosomes for linkage to schizophrenia in 126 small families and report a small positive LOD score for the marker DXS7, adjacent to the MAO locus at Xp11.4-11.3. Because of this, we have examined the DXS7 for linkage to schizophrenia using 17 pedigrees in which male-to-male transmission of schizophrenia was absent. Alleles at DXS7 were genotyped using the PCR and LOD scores calculated using five models of inheritance, including classical dominant, recessive and intermediate models. LOD scores were substantially negative for all models examined and analysis for linkage heterogeneity using the LOD2 method showed no significance. Analysis by the nonparametric affected sib-pair method likewise indicated no linkage. We conclude that DXS7 is not a major locus for schizophrenia in our collection of pedigrees. 29 refs., 1 tab.

  5. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  6. Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5'-phosphate biosynthesis.

    PubMed

    Belitsky, Boris R

    2004-02-01

    Bacillus subtilis synthesizes pyridoxal 5'-phosphate, the active form of vitamin B(6), by a poorly characterized pathway involving the yaaD and yaaE genes. The pdxS (yaaD) mutant was confirmed to be a strict B(6) auxotroph, but the pdxT (yaaE) mutant turned out to be a conditional auxotroph depending on the availability of ammonium in the growth medium. The PdxS and PdxT proteins copurified during affinity chromatography and apparently form a complex that has glutaminase activity. PdxS and PdxT appear to encode the synthase and glutaminase subunits, respectively, of a glutamine amidotransferase of as-yet-unknown specificity essential for B(6) biosynthesis.

  7. Biochemical genetics of the pentose phosphate cycle: human ribose 5-phosphate isomerase (RPI) and ribulose 5-phosphate 3-epimerase (RPE).

    PubMed

    Spencer, N; Hopkinson, D A

    1980-05-01

    1. Staining procedures are described for the detection after starch-gel electrophoresis of ribose-5-phosphate isomerase (RPI) and ribulose 5-phosphate 3-epimerase (RPE). 2. Both RPI and RPE were detected in all human tissues including red cells, lymphocytes and fibroblasts. 3. No evidence was found for more than one structural gene locus for either enzyme. 4. No allelic variants of either enzyme were found in erythrocyte lysates from over 200 unrelated individuals. 5. Preliminary data are presented which suggest that differences in tissue RPE isozyme patterns may be due to endogenous proteolytic activity. 6. Electrophoretic analysis of RPE and RPI isozyme patterns in extracts of man/mouse hybrid cells indicates that RPE is probably a dimer and RPI may also be polymeric.

  8. Linkage analysis of families with fragile-X mental retardation, using a novel RFLP marker (DXS 304).

    PubMed Central

    Dahl, N; Goonewardena, P; Malmgren, H; Gustavson, K H; Holmgren, G; Seemanova, E; Annerén, G; Flood, A; Pettersson, U

    1989-01-01

    A new polymorphic DNA marker U6.2, defining the locus DXS304, was recently isolated and mapped to the Xq27 region of the X chromosome. In the previous communication we describe a linkage study encompassing 16 fragile-X families and using U6.2 and five previously described polymorphic markers at Xq26-q28. One recombination event was observed between DXS304 and the fragile-X locus in 36 informative meioses. Combined with information from other reports, our results suggest the following order of the examined loci on Xq: cen-F9-DXS105-DXS98-FRAXA-DXS304-(DXS52-F8 -DXS15). The locus DXS304 is closely linked to FRAXA, giving a peak lod score of 5.86 at a corresponding recombination fraction of .00. On the basis of the present results, it is apparent that U6.2 is a useful probe for carrier and prenatal diagnosis in fragile-X families. PMID:2569270

  9. A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073.

    PubMed

    Mosberg, Joshua A; Yep, Alejandra; Meredith, Timothy C; Smith, Sara; Wang, Pan-Fen; Holler, Tod P; Mobley, Harry L T; Woodard, Ronald W

    2011-06-01

    Previous studies showed that deletion of genes c3405 to c3410 from PAI-metV, a genomic island from Escherichia coli CFT073, results in a strain that fails to compete with wild-type CFT073 after a transurethral cochallenge in mice and is deficient in the ability to independently colonize the mouse kidney. Our analysis of c3405 to c3410 suggests that these genes constitute an operon with a role in the internalization and utilization of an unknown carbohydrate. This operon is not found in E. coli K-12 but is present in a small number of pathogenic E. coli and Shigella boydii strains. One of the genes, c3406, encodes a protein with significant homology to the sugar isomerase domain of arabinose 5-phosphate isomerases but lacking the tandem cystathionine beta-synthase domains found in the other arabinose 5-phosphate isomerases of E. coli. We prepared recombinant c3406 protein, found it to possess arabinose 5-phosphate isomerase activity, and characterized this activity in detail. We also constructed a c3406 deletion mutant of E. coli CFT073 and demonstrated that this deletion mutant was still able to compete with wild-type CFT073 in a transurethral cochallenge in mice and could colonize the mouse kidney. These results demonstrate that the presence of c3406 is not essential for a pathogenic phenotype.

  10. Template-directed oligomerization of 3-isoadenosine 5'-phosphate

    NASA Technical Reports Server (NTRS)

    Hill, Aubrey R., Jr.; Orgel, Leslie E.; Kumar, Shiv; Leonard, Nelson J.

    1988-01-01

    Template-directed oligomerization of an activated derivative of 3-isoadenosine 5'-phosphate (piA) on polyuridylic acid was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3'-5'-linked oligomers while the reaction of ImpA gives only 2'-5'-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.

  11. Morton's foot and pyridoxal 5'-phosphate deficiency: genetically linked traits.

    PubMed

    Nichols, Trent W; Gaiteri, Christopher

    2014-12-01

    Vitamin B6 is an essential vitamin needed for many chemical reactions in the human body. It exists as several vitamins forms but pyridoxal 5'-phosphate (PLP) is the phosphorylated form needed for transamination, deamination, and decarboxylation. PLP is important in the production of neurotransmitters, acts as a Schiff base and is essential in the metabolism of homocysteine, a toxic amino acid involved in cardiovascular disease, stroke, thrombotic and Alzheimer's disease. This report announces the connection between a deficit of PLP with a genetically linked physical foot form known as the Morton's foot. Morton's foot has been associated with fibromyalgia/myofascial pain syndrome. Another gene mutation methylenetetrahydrofolate reductase (MTHFr) is now being recognized much commonly than previous with chronic fatigue, chronic Lyme diseases and as "the missing link" in other chronic diseases. PLP deficiency also plays a role in impaired glucose tolerance and may play a much bigger role in the obesity, diabetes, fatty liver and metabolic syndrome. Without the Schiff-base of PLP acting as an electron sink, storing electrons and dispensing them in the mitochondria, free radical damage occurs! The recognition that a phenotypical expression (Morton's foot) of a gene resulting in deficiency of an important cofactor enzyme pyridoxal 5'-phosphate will hopefully alert physicians and nutritionist to these phenomena. Supplementation with PLP, L5-MTHF, B12 and trimethylglycine should be used in those patients with hyperhomocysteinemia and/or MTHFR gene mutation. PMID:25441836

  12. Adenosine-5'-phosphate deaminase. A novel herbicide target.

    PubMed Central

    Dancer, J E; Hughes, R G; Lindell, S D

    1997-01-01

    The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool. PMID:9159944

  13. Purification and characterization of ribulose-5-phosphate kinase from spinach

    SciTech Connect

    Porter, M.A.; Milanez, S.; Stringer, C.D.; Hartman, F.C.

    1986-02-15

    An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.

  14. Inactive mutants of human pyridoxine 5'-phosphate oxidase: a possible role for a noncatalytic pyridoxal 5'-phosphate tight binding site.

    PubMed

    Ghatge, Mohini S; Karve, Sayali S; David, Tanya M S; Ahmed, Mostafa H; Musayev, Faik N; Cunningham, Kendra; Schirch, Verne; Safo, Martin K

    2016-05-01

    Pyridoxal 5'-phosphate (PLP) is a cofactor for many vitamin B6-requiring enzymes that are important for the synthesis of neurotransmitters. Pyridoxine 5'-phosphate oxidase (PNPO) is one of two enzymes that produce PLP. Some 16 known mutations in human PNPO (hPNPO), including R95C and R229W, lead to deficiency of PLP in the cell and have been shown to cause neonatal epileptic encephalopathy (NEE). This disorder has no effective treatment, and is often fatal unless treated with PLP. In this study, we show that R95C hPNPO exhibits a 15-fold reduction in affinity for the FMN cofactor, a 71-fold decrease in affinity for the substrate PNP, a 4.9-fold decrease in specific activity, and a 343-fold reduction in catalytic activity, compared to the wild-type enzyme. We have reported similar findings for R229W hPNPO. This report also shows that wild-type, R95C and R229W hPNPO bind PLP tightly at a noncatalytic site and transfer it to activate an apo-B6 enzyme into the catalytically active holo-form. We also show for the first time that hPNPO forms specific interactions with several B6 enzymes with dissociation constants ranging from 0.3 to 12.3 μm. Our results suggest a possible in vivo role for the tight binding of PLP in hPNPO, whether wild-type or variant, by protecting the very reactive PLP, and transferring this PLP directly to activate apo-B6 enzymes. PMID:27419045

  15. Chloroplast Activity and 3'phosphadenosine 5'phosphate Signaling Regulate Programmed Cell Death in Arabidopsis.

    PubMed

    Bruggeman, Quentin; Mazubert, Christelle; Prunier, Florence; Lugan, Raphaël; Chan, Kai Xun; Phua, Su Yin; Pogson, Barry James; Krieger-Liszkay, Anja; Delarue, Marianne; Benhamed, Moussa; Bergounioux, Catherine; Raynaud, Cécile

    2016-03-01

    Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response. PMID:26747283

  16. Chemistry and diversity of pyridoxal-5'-phosphate dependent enzymes.

    PubMed

    Phillips, Robert S

    2015-09-01

    Pyridoxal-5'-phosphate (PLP) is a versatile cofactor that enzymes use to catalyze a wide variety of reactions of amino acids, including transamination, decarboxylation, racemization, β- and γ-eliminations and substitutions, retro-aldol and Claisen reactions. These reactions depend on the ability of PLP to stabilize, to a varying degree, α-carbanionic intermediates. Furthermore, oxidative decarboxylations and rearrangements suggest that PLP can stabilize radical intermediates as well. The reaction mechanisms of two PLP-dependent enzymes are discussed, kynureninase and tyrosine phenol-lyase (TPL). Kynureninase catalyzes a retro-Claisen reaction of kynurenine to give anthranilate and alanine. The key step, hydration of the γ-carbonyl, is assisted by acid-base catalysis with the phosphate of the PLP, mediated by a conserved tyrosine, and an oxyanion hole. TPL catalyzes the reversible elimination of phenol, a poor leaving group, from l-tyrosine. In TPL, the Cβ-Cγ bond cleavage is accelerated by ground state strain from the bending of the substrate ring out of the plane with the Cβ-Cγ bond. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  17. Sequence analysis of two de novo mutation alleles at the DXS10011 locus.

    PubMed

    Tamura, Akiyoshi; Iwata, Misa; Takase, Izumi; Miyazaki, Tokiko; Matsui, Kiyoshi; Nishio, Hajime; Suzuki, Koichi

    2003-09-01

    We have detected two unusual alleles at the DXS10011 locus in two paternity trio cases. In one case, one allele of the daughter was found not to have been derived from the mother but the other allele was shared with the father. In the other case, the mother and the son shared no bands. Paternity in both cases was established using conventional polymorphic markers in addition to DNA markers (probabilities: >0.999999). Sequencing showed that the two de novo alleles of the children acquired a single unit (GAAA).

  18. Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate : new insights from structural and biochemical studies on human RPE.

    SciTech Connect

    Liang, W.; Ouyang, S.; Shaw, N.; Joachimiak, A.; Zhang, R.; Liu, Z.; Biosciences Division; Chinese Academy of Sciences

    2011-02-01

    The pentose phosphate pathway (PPP) confers protection against oxidative stress by supplying NADPH necessary for the regeneration of glutathione, which detoxifies H{sub 2}O{sub 2} into H{sub 2}O and O{sub 2}. RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe{sup 2+} for catalysis. Structures of the binary complexes of hRPE with D-ribulose 5-phosphate and D-xylulose 5-phosphate provide the first detailed molecular insights into the binding mode of physiological ligands and reveal an octahedrally coordinated Fe{sup 2+} ion buried deep inside the active site. Human RPE folds into a typical ({beta}/{alpha}){sub 8} triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. Two aspartic acids are well positioned to carry out the proton transfers in an acid-base type of reaction mechanism. Interestingly, mutating Ser-10 to alanine almost abolished the enzymatic activity, while L12A and M72A mutations resulted in an almost 50% decrease in the activity. The binary complexes of hRPE reported here will aid in the design of small molecules for modulating the activity of the enzyme and altering flux through the PPP.

  19. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    PubMed Central

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection. PMID:25568941

  20. Ribose 5-phosphate isomerase B knockdown compromises Trypanosoma brucei bloodstream form infectivity.

    PubMed

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection. PMID:25568941

  1. Ribose 5-phosphate isomerase B knockdown compromises Trypanosoma brucei bloodstream form infectivity.

    PubMed

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.

  2. Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system.

    PubMed

    Slabas, A R; Walker, D A

    1976-04-01

    Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO2 fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O2 evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected. The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient DL-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity. It is concluded that ribose 5-phosphate inhibits photosynthetic O2 evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.

  3. 1-deoxy-d-xylulose-5-phosphate reductoisomerases and method of use

    DOEpatents

    Croteau, Rodney B.; Lange, Bernd M.

    2001-01-01

    The present invention relates to isolated DNA sequences which code for the expression of plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein, such as the sequence presented in SEQ ID NO:1 which encodes a 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein from peppermint (Mentha x piperita). Additionally, the present invention relates to isolated plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, to modified host cells transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention.

  4. 1-deoxy-D-xylulose-5-phosphate reductoisomerases, and methods of use

    DOEpatents

    Croteau, Rodney B.; Lange, Bernd M.

    2002-07-16

    The present invention relates to isolated DNA sequences which code for the expression of plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein, such as the sequence presented in SEQ ID NO:1 which encodes a 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein from peppermint (Mentha x piperita). Additionally, the present invention relates to isolated plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, to modified host cells transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention.

  5. The structure of an archaeal ribose-5-phosphate isomerase from Methanocaldococcus jannaschii (MJ1603).

    PubMed

    Strange, Richard W; Antonyuk, Svetlana V; Ellis, Mark J; Bessho, Yoshitaka; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Hasnain, S Samar

    2009-12-01

    Ribose-5-phosphate isomerase is a ubiquitous intracellular enzyme of bacterial, plant and animal origin that is involved in the pentose phosphate cycle, an essential component of cellular carbohydrate metabolism. Specifically, the enzyme catalyses the reversible conversion of ribose 5-phosphate to ribulose 5-phosphate. The structure of ribose-5-phosphate isomerase from Methanocaldococcus jannaschii has been solved in space group P2(1) to 1.78 A resolution using molecular replacement with one homotetramer in the asymmetric unit and refined to an R factor of 14.8%. The active site in each subunit was occupied by two molecules of propylene glycol in different orientations, one of which corresponds to the location of the phosphate moiety and the other to the location of the furanose ring of the inhibitor.

  6. Purification and properties of D-ribulose-5-phosphate 3-epimerase from calf liver.

    PubMed

    Wood, T

    1979-10-11

    D-Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was purified 760-fold from calf liver by adsorption on DEAE-cellulose, chromatography on DEAE-Sephadex, chromatography on D-ribose 5-phosphate-Sepharose and gel filtration on Biogel P200. The purified enzyme of specific activity 617 units/mg was obtained in 28% yield and gave a single band on polyacrylamide gel electrophoresis. It had a molecular weight of 45 000 and appeared to contain two identical peptide chains of 22 900 daltons. The Km for D-ribulose 5-phosphate was 0.19 +/- 0.07 mM (S.E.). It was inhibited by reagents reacting with sulphydryl groups, by sulphate ion, and by D-deoxyribose 5-phosphate. The pH-stability and pH-activity curves were determined.

  7. Biosynthesis of riboflavin. Enzymatic formation of the xylene moiety from [14C]ribulose 5-phosphate.

    PubMed

    Nielsen, P; Neuberger, G; Floss, H G; Bacher, A

    1984-02-14

    We have studied the enzymatic formation of the xylene ring of riboflavin using cell extracts from the flavinogenic yeast Candida guilliermondii. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate could serve as substrates. In addition, a pentose phosphate or pentulose phosphate was required. Experiments with [14C]ribulose 5-phosphate gave evidence for the incorporation of the ribulose carbon atoms except C-4 into the xylene ring of the vitamin. PMID:6546684

  8. Synthesis and biological evaluation of arabinose 5-phosphate mimics modified at position five.

    PubMed

    Cipolla, Laura; Airoldi, Cristina; Sperandeo, Paola; Gianera, Serena; Polissi, Alessandra; Nicotra, Francesco; Gabrielli, Luca

    2014-05-01

    A set of new metabolically stable arabinose 5-phosphate analogues possessing phosphate mimetic groups at position 5 was synthesised. Their ability to interact with arabinose 5-phosphate isomerase from Pseudomonas aeruginosa was evaluated by STD-NMR studies. The synthesised compounds were also characterised for their activity in vivo on P. aeruginosa and Escherichia coli strains. Unfortunately, none of the synthesised compounds was able neither to bind API nor to inhibit bacterial growth.

  9. Host cells and methods for producing 1-deoxyxylulose 5-phosphate (DXP) and/or a DXP derived compound

    DOEpatents

    Kirby, James; Fortman, Jeffrey L.; Nishimoto, Minobu; Keasling, Jay D.

    2016-07-05

    The present invention provides for a genetically modified host cell capable of producing 1-deoxyxylulose 5-phosphate or 1-deoxy-D-xylulose 5-phosphate (DXP) (12), and optionally one or more DXP derived compounds, comprising: (a) a mutant RibB, or functional variant thereof, capable of catalyzing xylulose 5-phosphate and/or ribulose 5-phosphate to DXP, or (b) a YajO, or functional variant thereof, and a XylB, or functional variant thereof.

  10. The preparation of transketolase free from D-ribulose-5-phosphate 3-epimerase.

    PubMed

    Wood, T

    1981-06-15

    A procedure for the purification from Candida utilis of transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) free from D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was developed using acetone precipitation, elution from DEAE-cellulose, adsorption of epimerase by thiopropyl-Sepharose, and chromatography on D-ribose 5-phosphate-Sepharose and DEAE--Sephadex. The final product had a specific activity of 43 units/mg, a transketolase/epimerase activity ratio greater than 53 000 to 1, an apparent Km for D-xylulose 5-phosphate and D-ribose 5-phosphate of 77 and 430 microM, respectively, and ran as a single band using electrophoresis on polyacrylamide gel. It was inhibited by D-arabinose 5-phosphate and D-glucose 6-phosphate. During the purification by column chromatography, multiple forms of the enzyme were detected by gel electrophoresis but these gradually disappeared as the enzyme was further purified.

  11. ESR study of stable radicals in an irradiated single crystal of deoxyguanosine 5'-phosphate (Na salt)

    SciTech Connect

    Rakvin, B.; Herak, J.N.

    1981-11-01

    Three different radical species have been identified in an irradiated single crystal of deoxyguanosine 5'-phosphate at room temperature. The dominating species is a hydrogen-addition radical with spectroscopic characteristics similar to those of the N(7)-protonated H-addition radical in guanine/sup ./HCl. The well-resolved quartet ESR pattern is believed to belong to a radical in the sugar moiety formed by breakage of the furanose ring in the same manner as that reported earlier for deoxycytidine 5'-phosphate. The third species present is either a protonated anion or a deprotonated cation located in this six-member ring of the guanine base.

  12. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    PubMed

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.

  13. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    PubMed

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks. PMID:27106120

  14. A new human gene (DXS1357E) with ubiquitous expression, located in Xq28 adjacent to the adrenoleukodystrophy gene

    SciTech Connect

    Mosser, J.; Sarde, C.O.; Vicaire, S.

    1994-07-15

    The authors have isolated a new human gene (DXS1357E; laboratory name: CDM) localized in Xq28. This gene is transcribed from the same CpG island as the adrenoleukodystrophy gene (ALD) and is oriented in the opposite direction. It encodes a 1.5-kb transcript that exhibits ubiquitous expression and contains a single open reading frame. The 246 deduced amino acid sequence suggests the presence of membrane-associated segments and a weak similarity with the rod-like tail portion of heavy chain myosins from different species. The DXS1357E gene may be a candidate for one of the many diseases mapping to this region. A preliminary analysis did not show rearrangements of the gene in 19 independent patients with Emery-Dreifuss muscular dystrophy. 21 refs., 2 figs.

  15. Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase.

    PubMed

    Kondo, Hiroki; Nakamura, Yoriko; Dong, Yi-Xin; Nikawa, Jun-ichi; Sueda, Shinji

    2004-04-01

    To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine. The enzymic activity of the Arg189-->Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.

  16. Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.

    PubMed

    Cech, David; Wang, Pan Fen; Holler, Tod P; Woodard, Ronald W

    2014-08-01

    Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and D-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-D-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5'-monophospho-3-deoxy-D-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a Ki of 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships.

  17. Concerted Proton Transfer Mechanism of Clostridium thermocellum Ribose-5-phosphate Isomerase

    PubMed Central

    Wang, Jun; Yang, Weitao

    2013-01-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to RpiB family, has recently been employed to the industrial production of rare sugars because of it fast reactions kinetics and narrow substrate specificity. It is known this enzyme adopts proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of substrate to initiate the isomerization reaction and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracts the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of RpiB family and should guide the experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines. PMID:23875675

  18. Concerted proton transfer mechanism of Clostridium thermocellum ribose-5-phosphate isomerase.

    PubMed

    Wang, Jun; Yang, Weitao

    2013-08-15

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to the RpiB family, has recently been employed in the industrial production of rare sugars because of its fast reaction kinetics and narrow substrate specificity. It is known that this enzyme adopts a proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of the substrate to initiate the isomerization reaction, and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family, performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracting the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of the RpiB family and should guide experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines.

  19. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids.

    PubMed

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-05-27

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes' replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids.

  20. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids

    PubMed Central

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes’ replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  1. Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications

    PubMed Central

    Chiu, Hsiu-Ju; Grant, Joanna C.; Farr, Carol L.; Jaroszewski, Lukasz; Knuth, Mark W.; Miller, Mitchell D.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2014-01-01

    The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5′-monophosphate-3-deoxy-d-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections. PMID:25286848

  2. Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications.

    PubMed

    Chiu, Hsiu Ju; Grant, Joanna C; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2014-10-01

    The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.

  3. Optic atrophy in Leber hereditary optic neuroretinopathy is probably determined by an X-chromosomal gene closely linked to DXS7.

    PubMed Central

    Vilkki, J; Ott, J; Savontaus, M L; Aula, P; Nikoskelainen, E K

    1991-01-01

    Leber hereditary optic neuroretinopathy (LHON) is a maternally inherited disease, probably transmitted by mutations in mtDNA. The variation in the clinical expression of the disease among family members has remained unexplained, but pedigree data suggest an involvement of an X-chromosomal factor. We have studied genetic linkage of the liability to develop optic atrophy to 15 polymorphic markers on the X chromosome in six pedigrees with LHON. The results show evidence of linkage to the locus DXS7 on the proximal Xp. Tight linkage to the other marker loci was excluded. Multipoint linkage analysis placed the liability locus at DXS7 with a maximum lod score (Zmax) of 2.48 at a recombination fraction (theta) of .0 and with a Zmax - 1 support interval theta = .09 distal to theta = .07 proximal of DXS7. No evidence of heterogeneity was found among different types of families, with or without a known mtDNA mutation associated with LHON. PMID:1998335

  4. Acetate selective fluorescent turn-on sensors derived using vitamin B6 cofactor pyridoxal-5-phosphate

    NASA Astrophysics Data System (ADS)

    Sharma, Darshna; Kuba, Aman; Thomas, Rini; Ashok Kumar, S. K.; Kuwar, Anil; Choi, Heung-Jin; Sahoo, Suban K.

    2016-03-01

    Two new Schiff base receptors have been synthesized by condensation of pyridoxal-5-phosphate with 2-aminophenol (L1) or aniline (L2). In DMSO, the receptors showed both chromogenic and 'turn-on' fluorescence responses selectively in the presence of AcO- and F-. However, in mixed DMSO-H2O medium, the receptors showed AcO- selective 'turn-on' fluorescence without any interference from other tested anions including F-. The detection limit for AcO- was found to be 7.37 μM and 22.9 μM using the receptors L1 and L2, respectively.

  5. Identification and characterization of a novel Ribose 5-phosphate isomerase B from Leishmania donovani.

    PubMed

    Kaur, Preet Kamal; Dinesh, Neeradi; Soumya, Neelagiri; Babu, Neerupudi Kishore; Singh, Sushma

    2012-04-27

    Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519 bp and encodes for a putative 172 amino acid protein with a molecular mass of ∼19 kDa. An ∼19 kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ∼19 kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis-Menten kinetics utilizing R5P as the substrate with a K(m) value of 2.4±0.6 mM and K(cat) value of 30±5.2 s(-1). Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target.

  6. Structure of Escherichia coli Ribose-5-Phosphate Isomerase: A Ubiquitous Enzyme of the Pentose Phosphate Pathway and the Calvin Cycle

    PubMed Central

    Zhang, Rong-guang; Andersson, C. Evalena; Savchenko, Alexei; Skarina, Tatiana; Evdokimova, Elena; Beasley, Steven; Arrowsmith, Cheryl H.; Edwards, Aled M.; Joachimiak, Andrzej; Mowbray, Sherry L.

    2009-01-01

    Summary Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 Å resolution (R factor 22.4%, Rfree 23.7%). RpiA exhibits an α/β/(α/β)/β/α fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 Å resolution. A mechanism for acid-base catalysis is proposed. PMID:12517338

  7. Structure of escherichia coli ribose-5-phosphate isomerase : a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle.

    SciTech Connect

    Zhang, R.; Andersson, C. E.; Savchenko, A.; Skarina, T.; Evdokimova, E.; Beasley, S.; Arrowsmith, C. H.; Edwards, A.; Joachimiak, A.; Mowbray, S. L.; Biosciences Division; Uppsala Univ.; Univ. Health Network; Univ. of Toronto; Swedish Univ. of Agricultural Sciences

    2003-01-01

    Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 Angstroms resolution (R factor 22.4%, R{sub free} 23.7%). RpiA exhibits an {alpha}/{beta}/({alpha}/{beta})/{beta}/{alpha} fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution. A mechanism for acid-base catalysis is proposed.

  8. Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

    SciTech Connect

    Abell, L.M.; O'Leary, M.H.

    1988-08-09

    The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9770 +/- 0.0021, a carbon isotope effect k/sup 12//k/sup 13/ = 1.0308 +/- 0.0006, and a carbon isotope effect for L-(..cap alpha..-/sup 2/H)histidine of 1.0333 +/- 0.0001 at pH 6.3, 37/sup 0/C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli, the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.

  9. Novel substrates of a ribose-5-phosphate isomerase from Clostridium thermocellum.

    PubMed

    Yoon, Ran-Young; Yeom, Soo-Jin; Kim, Hye-Jung; Oh, Deok-Kun

    2009-01-01

    A substrate specificity study of the recombinant D-ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum was performed. Among all aldopentoses and aldohexoses, the RpiB enzyme displayed activity with L-talose, D-ribose, D-allose, L-allose, L-ribose, and D-talose in decreasing order. The products released were L-tagatose, D-ribulose, D-psicose, L-psicose, L-ribulose, and D-tagatose, respectively. The enzyme showed specificity for aldose substrates possessing hydroxyl groups oriented in the same direction at the C2, C3, and C4 positions. Molecular modeling of the enzyme suggests that the novel substrate specificity may be explained by substrate interactions with residues Tyr42, His98, and His9, which interact with the hydroxyl groups of C2, C3, and C4, respectively, oriented in the same direction. L-Talose and D-ribulose exhibited the highest activity among the aldoses and ketoses, respectively. Ribose 5-phosphate isomerase catalyzed the conversion of L-talose to L-tagatose with an 89% conversion yield after approximately 90 min, while D-ribulose was converted to D-ribose with a 38% conversion yield.

  10. Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase

    SciTech Connect

    Omnaas, J.; Porter, M.A.; Hartman, F.C.

    1985-02-01

    Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 m s at 2C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with UC-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with UC-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence, two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. The reactive sulfhydryl groups are likely to be those generated by reduction of a disulfide during activation. 20 references, 3 figures, 2 tables.

  11. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    SciTech Connect

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  12. Identification of GutQ from Escherichia coli as a D-arabinose 5-phosphate isomerase.

    PubMed

    Meredith, Timothy C; Woodard, Ronald W

    2005-10-01

    The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol D-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a D-arabinose 5-phosphate isomerase (API) from the 3-deoxy-D-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between D-ribulose 5-phosphate (Ru5P) and D-arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(deltakdsD)] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(deltagutQ)], suggesting that GutQ is not directly involved in d-glucitol catabolism. The conditional mutant TCM15[(deltagutQdeltakdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d-glucitol and there is no readily apparent link between D-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.

  13. 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. PMID:25568319

  14. Pseudouridine synthases.

    PubMed

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  15. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates.

    PubMed

    Pichlmair, Andreas; Schulz, Oliver; Tan, Choon Ping; Näslund, Tanja I; Liljeström, Peter; Weber, Friedemann; Reis e Sousa, Caetano

    2006-11-10

    Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

  16. Ribose-5-phosphate biosynthesis in Methanocaldococcus jannaschii occurs in the absence of a pentose-phosphate pathway.

    PubMed

    Grochowski, Laura L; Xu, Huimin; White, Robert H

    2005-11-01

    Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-2H2]- and [U-13C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of D-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the D-ara-bino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from D-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.

  17. Functional analysis of the final steps of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing.

    PubMed

    Page, Jonathan E; Hause, Gerd; Raschke, Maja; Gao, Wenyun; Schmidt, Jürgen; Zenk, Meinhart H; Kutchan, Toni M

    2004-04-01

    Isoprenoid biosynthesis in plant plastids occurs via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. We used tobacco rattle virus (TRV) to posttranscriptionally silence the expression of the last two enzymes of this pathway, the IspG-encoded (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and the IspH-encoded isopentenyl/dimethylallyl diphosphate synthase (IDDS), as well as isopentenyl/dimethylallyl diphosphate isomerase (IDI), the enzyme that interconverts IPP and DMAPP. TRV-IspG and TRV-IspH infected Nicotiana benthamiana plants had albino leaves that contained less than 4% of the chlorophyll and carotenoid pigments of control leaves. We applied [(13)C]DXP and [(14)C]DXP to silenced leaves and found that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate accumulated in plants blocked at HDS while DXP, (E)-4-hydroxy-3-methylbut-2-enyl phosphate and (E)-2-methylbut-2-ene-1,4-diol accumulated in IDDS-blocked plants. Albino leaves from IspG- and IspH-silenced plants displayed a disorganized palisade mesophyll, reduced cuticle, fewer plastids, and disrupted thylakoid membranes. These findings demonstrate the participation of HDS and IDDS in the DXP pathway in plants, and support the view that plastid isoprenoid biosynthesis is metabolically and physically segregated from the mevalonate pathway. IDI-silenced plants had mottled white-pale green leaves with disrupted tissue and plastid structure, and showed an 80% reduction in pigments compared to controls. IPP pyrophosphatase activity was higher in chloroplasts isolated from IDI-silenced plants than in control plant chloroplasts. We suggest that a low level of isoprenoid biosynthesis via the DXP pathway can occur without IDI but that this enzyme is required for full function of the DXP pathway.

  18. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    PubMed Central

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation. PMID:23650515

  19. Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus.

    PubMed

    Han, Mei; Heppel, Simon C; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

  20. Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis.

    PubMed

    Cao, Thinh-Phat; Kim, Joong-Su; Woo, Mi-Hee; Choi, Jin Myung; Jun, Youngsoo; Lee, Kun Ho; Lee, Sung Haeng

    2016-04-01

    2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/β)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and β8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.

  1. First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.

    PubMed

    Ohshida, Tatsuya; Hayashi, Junji; Satomura, Takenori; Kawakami, Ryushi; Ohshima, Toshihisa; Sakuraba, Haruhiko

    2016-10-01

    2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA. PMID:27215670

  2. First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.

    PubMed

    Ohshida, Tatsuya; Hayashi, Junji; Satomura, Takenori; Kawakami, Ryushi; Ohshima, Toshihisa; Sakuraba, Haruhiko

    2016-10-01

    2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA.

  3. Inhibition and site modification of human hepatitis B virus DNA polymerase by pyridoxal 5'-phosphate

    SciTech Connect

    Oh, S.H.; Park, Y.H.; Kim, I.S.; Woo, K.

    1987-05-01

    Pyridoxal 5'-phosphate(PLP) modification of human hepatitis B virus (H3V) DNA polymerase was attempted in order to characterize the nature of the enzyme. Dane particle cores isolated from serum of a chronic HBV carrier by sucrose density gradient centrifugation contained DNA polymerase activity, and the enzyme activity was inhibited specifically by PLP in noncompetitive fashion with respective to dNTP. Kinetic study indicates that HBV DNA polymerase has a Km of 0.31..mu..M for dTTP and an apparent Ki of 2mM for PLP. Sodium borohydride reduction of PLP-HEV core particles caused almost complete inhibition of HBV DNA polymerase activity. Reduction of PLP-HBV core particles by /sup 3/H labeled NaBH4 followed by SDS polyacrylamide gel electrophoresis was carried out, and the fluorography of the SDS polyacrylamide gel revealed 3 major bands corresponding to molecular weights of 21,000, 80,000 and > 100,000. Dane particle associated DNA polymerase inhibition by PLP is mediated through Schiff's base formation with a free amino group present at catalytic site of the enzyme. A core protein having an approximate molecular weight of 80,000 is considered as HBV DNA polymerase.

  4. Molecular evolution of the bacterial pseudouridine-5'-phosphate glycosidase protein family.

    PubMed

    Thapa, Keshav; Oja, Terhi; Metsä-Ketelä, Mikko

    2014-10-01

    Pseudouridine is a noncanonical C-nucleoside commonly present in RNA, which is not metabolized in mammals, but can be recycled by the unique enzyme family of bacterial pseudouridine glycosidases such as YeiN from Escherichia coli. Here, we present rigorous bioinformatic and biochemical analyses of the protein family in order to find sequences that might code for nonpseudouridine glycosidase activities. To date, the only other function reported for the enzyme family occurs during the biosynthesis of the antibiotic alnumycin A in Streptomyces species, where AlnA functions as an unusual C-glycosynthase. Bioinformatics analysis of 755 protein sequences identified one group of sequences that were unlikely to harbour pseudouridine glycosidase activities. This observation was confirmed in vitro with one representative protein, IdgA from Streptomyces albus, which was unable to synthesize pseudouridine monophosphate, but was able to attach d-ribose-5-phosphate to juglone. Furthermore, our analyses provide evidence for horizontal gene transfer of pseudouridine glycosidases that may have occurred in Streptomyces and Doria species. Inspection of the genomic loci in the vicinity of pseudouridine glycosidases revealed that in 77% of the strains a kinase gene putatively involved in the phosphorylation of pseudouridine was found nearby, whereas the sequences encoding nonpseudouridine glycosidases coexisted with a phosphatase of the haloacid dehalogenase enzyme family. The investigation suggested that these unknown sequences might be involved in the biosynthesis of soluble blue pigments because of the presence of genes homologous to nonribosomal peptide synthetases.

  5. Metabolite Profiling of Plastidial Deoxyxylulose-5-Phosphate Pathway Intermediates by Liquid Chromatography and Mass Spectrometry

    PubMed Central

    Baidoo, Edward E.K.; Xiao, Yanmei; Dehesh, Katayoon; Keasling, Jay D.

    2016-01-01

    Metabolite profiling is a powerful tool that enhances our understanding of complex regulatory processes and extends to the comparative analysis of plant gene function. However, at present, there are relatively few examples of metabolite profiling being used to characterize the regulatory aspects of the plastidial deoxyxylulose-5-phosphate (DXP) pathway in plants. Since the DXP pathway is one of two pathways in plants that are essential for isoprenoid biosynthesis, it is imperative that robust analytical methods be employed for the characterization of this metabolic pathway. Recently, liquid chromatography-mass spectrometry (LC-MS), in conjunction with traditional molecular biology approaches, established that the DXP pathway metabolite, methylerythritol cyclodiphosphate (MEcPP), previously known solely as an intermediate in the isoprenoid biosynthetic pathway, is a stress sensor that communicates environmental perturbations sensed by plastids to the nucleus, a process referred to as retrograde signaling. In this chapter, we describe two LC-MS methods from this study that can be broadly used to characterize DXP pathway intermediates. PMID:24777790

  6. Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase alpha and epsilon.

    PubMed

    Mizushina, Yoshiyuki; Xu, Xianai; Matsubara, Kiminori; Murakami, Chikako; Kuriyama, Isoko; Oshige, Masahiko; Takemura, Masaharu; Kato, Norihisa; Yoshida, Hiromi; Sakaguchi, Kengo

    2003-12-26

    Vitamin B(6) compounds such as pyridoxal 5(')-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which reportedly have anti-angiogenic and anti-cancer effects, were thought to be inhibitors of some types of eukaryotic DNA polymerases. PL moderately inhibited only the activities of calf DNA polymerase alpha (pol alpha), while PN and PM had no inhibitory effects on any of the polymerases tested. On the other hand, PLP, a phosphated form of PL, was potentially a strong inhibitor of pol alpha and epsilon from phylogenetic-wide organisms including mammals, fish, insects, plants, and protists. PLP did not suppress the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as deoxyribonuclease I. For pol alpha and epsilon, PLP acted non-competitively with the DNA template-primer and competitively with the nucleotide substrate. Since PL was converted to PLP in vivo after being incorporated into human cancer cells, the anti-angiogenic and anti-cancer effects caused by PL must have been caused by the inhibition of pol alpha and epsilon activities after conversion to PLP.

  7. The localization of a gene causing X-linked cleft palate and ankyloglossia (CPX) in an Icelandic kindred is between DXS326 and DXYS1X.

    PubMed

    Stanier, P; Forbes, S A; Arnason, A; Bjornsson, A; Sveinbjornsdottir, E; Williamson, R; Moore, G

    1993-09-01

    The locus responsible for X-linked, nonsyndromic cleft palate and/or ankyloglossia (CPX) has previously been mapped to the proximal long arm of the human X chromosome between Xq21.31 and q21.33 in an Icelandic kindred. We have extended these studies by analyzing an additional 14 informative markers in the family as well as including several newly investigated family members. Recombination analysis indicates that the CPX locus is more proximal than previously thought, within the interval Xq21.1-q21.31. Two recombinants place DXYS1X as the distal flanking marker, while one recombinant defines DXS326 as the proximal flanking marker, an interval of less than 5 cM. Each of the flanking markers recombines with the CPX locus, giving 2-point lod scores of Zmax = 4.16 at theta = 0.08 (DXS326) and Zmax = 5.80 at theta = 0.06 (DXYS1X).

  8. Development of user-friendly functional molecular markers for VvDXS gene conferring muscat flavor in grapevine.

    PubMed

    Emanuelli, F; Sordo, M; Lorenzi, S; Battilana, J; Grando, M S

    2014-01-01

    High fruit and wine quality combined with good climatic adaptation and disease resistance are essential objectives of grape breeding. While several molecular markers are available for pyramiding resistance to fungal pathogens, molecular tools for predicting fruit composition are still scarce. Muscat flavor, caused by the accumulation of monoterpenoids in the berry, is an important target trait for breeding, sought after in both table grapes and wine. Four missense mutations in the VvDXS gene in grape germplasm have been shown to be tightly linked to muscat flavor. Here we present highly reproducible and breeder-friendly functional markers for each of the targeted polymorphisms developed by using either the multiplexed minisequencing SNaPshot™ method, the high-resolution melting (HRM) assay or the cleaved amplified polymorphic sequence system. A total of 242 grapevine accessions were analyzed to optimize these different genotyping methods and to provide allele-specific markers for accurate selection of muscat flavor at early stages of grape breeding programs. The HRM and the minisequencing SNaPshot multiplex assays allow for high-throughput automated screening and are suitable for large-scale breeding programs and germplasm characterization. PMID:24482604

  9. Quantitative effect and regulatory function of cyclic adenosine 5'-phosphate in Escherichia coli.

    PubMed

    Narang, Atul

    2009-09-01

    Cyclic adenosine 5'-phosphate (cAMP) is a global regulator of gene expression in Escherichia coli. Despite decades of intensive study, the quantitative effect and regulatory function of cAMP remain the subjects of considerable debate. Here, we analyse the data in the literature to show that: (a) In carbon-limited cultures (including cultures limited by glucose), cAMP is at near-saturation levels with respect to expression of several catabolic promoters (including lac, ara and gal). It follows that cAMP receptor protein (CRP) cAMP-mediated regulation cannot account for the strong repression of these operons in the presence of glucose. (b) The cAMP levels in carbon-excess cultures are substantially lower than those observed in carbon-limited cultures under these conditions, the expression of catabolic promoters is very sensitive to variation of cAMP levels. (c)=CRPcAMP invariably activates the expression of catabolic promoters, but it appears to inhibit the expression of anabolic promoters. (d) These results suggest that the physiological function of cAMP is to maintain homeostatic energy levels. In carbon-limited cultures, growth is limited by the supply of energy; the cAMP levels therefore increase to enhance energy accumulation by activating the catabolic promoters and inhibiting the anabolic promoters. Conversely, in carbonexcess cultures, characterized by the availability of excess energy, the cAMP levels decrease in order to depress energy accumulation by inhibiting the catabolic promoters and activating the anabolic promoters. PMID:19805906

  10. Competitive inhibitors of Mycobacterium tuberculosis ribose-5-phosphate isomerase B reveal new information about the reaction mechanism.

    PubMed

    Roos, Annette K; Burgos, Emmanuel; Ericsson, Daniel J; Salmon, Laurent; Mowbray, Sherry L

    2005-02-25

    Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (K(m) 57 microm) and 4-phospho-d-erythronate (K(i) 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.

  11. Crystal structure of Clostridium thermocellum ribose-5-phosphate isomerase B reveals properties critical for fast enzyme kinetics.

    PubMed

    Jung, Junho; Kim, Jin-Kwang; Yeom, Soo-Jin; Ahn, Yeh-Jin; Oh, Deok-Kun; Kang, Lin-Woo

    2011-04-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of D-ribose 5-phosphate (R5P) to D-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, D-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-Å resolution or higher, of CtRpi alone and bound to its substrates, R5P, D-ribose, and D-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi.

  12. Contiguous Deletion of the X-Linked Adrenoleukodystrophy Gene (ABCD1) and DXS1357E: A Novel Neonatal Phenotype Similar to Peroxisomal Biogenesis Disorders

    PubMed Central

    Corzo, Deyanira; Gibson, William; Johnson, Kisha; Mitchell, Grant; LePage, Guy; Cox, Gerald F.; Casey, Robin; Zeiss, Carolyn; Tyson, Heidi; Cutting, Garry R.; Raymond, Gerald V.; Smith, Kirby D.; Watkins, Paul A.; Moser, Ann B.; Moser, Hugo W.; Steinberg, Steven J.

    2002-01-01

    X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs to the ATP-binding cassette–transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-chain fatty acids (VLCFA) (carbon length >22). Childhood cerebral X-ALD is the most devastating form of the disease. These children have the earliest onset (age 7.2 ± 1.7 years) among the clinical phenotypes for ABCD1 mutations, but onset does not occur at <3 years of age. Individuals with either peroxisomal biogenesis disorders (PBD) or single-enzyme deficiencies (SED) in the peroxisomal β-oxidation pathway—disorders such as acyl CoA oxidase deficiency and bifunctional protein deficiency—also accumulate VLCFA, but they present during the neonatal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene, DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We propose that the term “contiguous ABCD1 DXS1357E deletion syndrome” (CADDS) be used to identify this new contiguous-gene syndrome. The three patients with CADDS who are described here have important implications for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an autosomal recessive PBD or SED PMID:11992258

  13. 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

  14. Deficiency in a cytosolic ribose-5-phosphate isomerase causes chloroplast dysfunction, late flowering and premature cell death in Arabidopsis.

    PubMed

    Xiong, Yuqing; DeFraia, Christopher; Williams, Donna; Zhang, Xudong; Mou, Zhonglin

    2009-11-01

    The oxidative pentose phosphate pathway (oxPPP) is part of central metabolism, consisting of two distinct phases: the oxidative phase and the non-oxidative phase. The non-oxidative phase of the oxPPP generates carbon skeletons for the synthesis of nucleotides, aromatic amino acids, phenylpropanoids and their derivatives, which are essential for plant growth and development. However, it is not well understood how the non-oxidative phase of the oxPPP contributes to plant growth and development. Here, we report the characterization of Arabidopsis T-DNA knockout mutants of the RPI2 gene (At2g01290), which encodes a cytosolic ribose-5-phosphate isomerase (RPI) that catalyzes the reversible interconversion of ribulose-5-phosphate and ribose-5-phosphate in the non-oxidative phase of the oxPPP. Although recombinant Arabidopsis RPI2 protein exhibits marked RPI enzymatic activity, knockout of the RPI2 gene does not significantly change the total RPI activity in the mutant plants. Interestingly, knockout of RPI2 interferes with chloroplast structure and decreases chloroplast photosynthetic capacity. The rpi2 mutants accumulate less starch in the leaves and flower significantly later than wild-type when grown under short-day conditions. Furthermore, the rpi2 mutants display premature cell death in the leaves when grown at an above-normal temperature (26 degrees C). These results demonstrate that a deficiency in the non-oxidative phase of the cytosolic oxPPP has pleiotropic effects on plant growth and development and causes premature cell death.

  15. Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Moriyama, Takashi; Tajima, Naoyuki; Sekine, Kohsuke; Sato, Naoki

    2015-01-01

    Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

  16. Characterization of ribose-5-phosphate isomerase converting D-psicose to D-allose from Thermotoga lettingae TMO.

    PubMed

    Feng, Zaiping; Mu, Wanmeng; Jiang, Bo

    2013-05-01

    The gene coding for ribose-5-phosphate isomerase (Rpi) from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant enzyme was purified by Ni-affinity chromatography. It converted D-psicose to D-allose maximally at 75 °C and pH 8.0 with a 32 % conversion yield. The k m, turnover number (k cat), and catalytic efficiency (k cat k m (-1) ) for substrate D-psicose were 64 mM, 6.98 min(-1) and 0.11 mM(-1) min(-1) respectively.

  17. The localization of a gene causing X-linked cleft palate and ankyloglossia (CPX) in an Icelandic kindred is between DXS326 and DXYS1X

    SciTech Connect

    Stainer, P.; Forbes, S.A.; Moore, G. ); Arnason, A.; Sveinbjornsdottir, E. ); Bjornsson, A. ); Williamson, R. )

    1993-09-01

    The locus responsible for X-linked, nonsyndromic cleft palate and/or ankyloglossia (CPX) has previously been mapped to the proximal long arm of the human X chromosome between Xq21.31 and q21.33 in an Icelandic kindred. The authors have extended these studies by analyzing an additional 14 informative markers in the family as well as including several newly investigated family members. Recombination analysis indicates that the CPX locus is more proximal than previously thought, within the interval Xq21.1-q21.31. Two recombinants place DXYS1X as the distal flanking marker, while one recombinant defines DXS326 as the proximal flanking marker, an interval of less than 5 cM. Each of the flanking markers recombines with the CPX locus, giving 2-point lod scores of Z[sub max] = 4.16 at [theta] = 0.08 (DXS326) and Z[sub max] = 5.80 at [theta] = 0.06 (DXYS1X). 35 refs., 3 figs., 2 tabs.

  18. Analysis of FMR1 (CGG)(n) alleles and DXS548-FRAXAC1 haplotypes in three European circumpolar populations: traces of genetic relationship with Asia.

    PubMed

    Larsen, L A; Vuust, J; Nystad, M; Evseeva, I; Van Ghelue, M; Tranebjaerg, L

    2001-09-01

    Fragile X syndrome, the most common form of inherited mental retardation, is caused by expansion of a (CGG)(n) repeat located in the FMR1 gene. The molecular factors involved in the mutation process from stable (CGG)(n) alleles towards unstable alleles are largely unknown, although family transmission studies and population studies have suggested that loss of AGG interruptions in the (CGG)(n) repeat is essential. We have analysed the AGG interspersion pattern of the FMR1 (CGG)(n) repeat and the haplotype distribution of closely located microsatellite markers DXS548 and FRAXAC1, in three circumarctic populations: Norwegians, Nenets and Saami. The data confirm the conservation, reported in all human populations studied so far, of an AGG interruption for each 9-10 CGG and support the stabilising effect of AGG interruptions. The data also indicate the existence of chromosomes of Asian origin in the Saami and Nenets population, thereby confirming a genetic relationship between Northern Europe and Asia. DXS548-FRAXAC1 haplotype frequencies were compared between 24 Norwegian fragile X males and 119 normal males. Significant linkage disequilibrium were found between the fragile X mutation and haplotype 6-4 and between normal (CGG)(n) alleles and haplotype 7-3.

  19. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

    PubMed

    Saladié, Montserrat; Wright, Louwrance P; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A

    2014-09-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties.

  20. Competitive inhibitors of yeast phosphoglucose isomerase: synthesis and evaluation of new types of phosphorylated sugars from the synthon D-arabinolactone-5-phosphate.

    PubMed

    Hardré, R; Salmon, L

    1999-05-31

    Designed as competitive inhibitors of the isomerization reaction catalyzed by the potential chemotherapeutic target phosphoglucose isomerases (PGI), D-arabinonamide-5-phosphate and D-arabinohydrazine-5-phosphate were synthesized and fully characterized. These new types of phosphorylated sugar derivatives were easily and efficiently obtained in a one-step procedure from the promising synthon D-arabinono-1,4-lactone 5-phosphate. These two compounds proved to be new good competitive inhibitors of yeast PGI with the substrate D-fructose-6-phosphate, though not as strong as D-arabinohydroxamic acid-5-phosphate. Overall, our results are in accord with the postulated 1,2-cis-enediolate species as a probable high-energy intermediate of the PGI-catalyzed reaction.

  1. Reconstitution of the pyridoxal 5'-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis.

    PubMed

    Beattie, Ashley E; Clarke, David J; Wadsworth, John M; Lowther, Jonathan; Sin, Ho-Lam; Campopiano, Dominic J

    2013-08-14

    The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) is required for de novo sphingolipid biosynthesis. A previous study revealed a novel and unexpected interaction between the hydroxyl group of the l-serine substrate and the 5'-phosphate group of PLP. By using pyridoxal (PL), the dephosphorylated analogue of vitamin B6, we show here that this interaction is important for substrate specificity and optimal catalytic efficiency.

  2. Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

    PubMed

    Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

    2016-08-10

    Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization.

  3. Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway.

    PubMed

    Williams, J F; Blackmore, P F

    1983-01-01

    1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in

  4. Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control[C][W

    PubMed Central

    Pulido, Pablo; Toledo-Ortiz, Gabriela; Phillips, Michael A.; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2013-01-01

    Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress. PMID:24104567

  5. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

    PubMed

    Simpson, Kevin; Quiroz, Luis F; Rodriguez-Concepción, Manuel; Stange, Claudia R

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products. PMID:27630663

  6. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products. PMID:27630663

  7. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  8. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

    PubMed

    Simpson, Kevin; Quiroz, Luis F; Rodriguez-Concepción, Manuel; Stange, Claudia R

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  9. Characterization of ribose-5-phosphate isomerase of Clostridium thermocellum producing D-allose from D-psicose.

    PubMed

    Park, Chang-Su; Yeom, Soo-Jin; Kim, Hye-Jung; Lee, Sook-Hee; Lee, Jung-Kul; Kim, Seon-Won; Oh, Deok-Kun

    2007-09-01

    The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted D-psicose into D-allose but it did not convert D-xylose, L-rhamnose, D-altrose or D-galactose. The production of D-allose by RpiB was maximal at pH 7.5 and 65 degrees C for 30 min. The half-lives of the enzyme at 50 degrees C and 65 degrees C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50 degrees C, 165 g D-allose l(-1 ) was produced without by-products from 500 g D-psicose l(-1) after 6 h.

  10. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  11. Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera.

    PubMed

    Gupta, Parul; Agarwal, Aditya Vikram; Akhtar, Nehal; Sangwan, Rajender Singh; Singh, Surya Pratap; Trivedi, Prabodh Kumar

    2013-02-01

    Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-D-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon-intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.

  12. A stable isotope dilution LC-ESI-MS/MS method for the quantification of pyridoxal-5'-phosphate in whole blood.

    PubMed

    van Zelst, Bertrand D; de Jonge, Robert

    2012-08-15

    Vitamin B6 is a cofactor in numerous biologic processes that include gluconeogenesis, neurotransmitter synthesis and amino acid metabolism. The aim of this study was to develop a method to measure the concentration of the biologically active form of vitamin B6 (pyridoxal-5'-phosphate, PLP) in whole blood with stable isotope dilution LC-ESI-MS/MS and compare this new procedure with an established HPLC method based on derivatization of pyridoxal-5'-phosphate. 50 μl of stable isotope (PLP-d3) was added to 250 μl of sample, followed by deproteinization with 10% trichloroacetic acid. After centrifugation, 20 μl of the supernatant was injected into the LC-ESI-MS/MS. Reversed phase chromatography was performed on a UPLC system, using a Waters™ Symmetry C18 column, with a gradient of 0.1% formic acid in methanol. PLP was measured on a tandem MS with a mass transition of 247.8>149.8 in the positive ion mode with a collision energy of 14 eV. The chromatographic run lasted 4 min. The method was linear from 4 to 8000 nmol/l. The intra-day and inter-day precision ranged between 1.7-2.8% and 3.0-4.1%, respectively. The mean absolute matrix-effect was 99.3% [97-102%]. The relative matrix-effect was 98.8%. The mean recovery was 98% [89-103%]. The lower limit of quantification was 4 nmol/l. The comparison of the LC-ESI-MS/MS method with our current HPLC method yielded the following equation: LC-ESI-MS/MS=1.11 [confidence interval, CI: 1.03-1.20] × HPLC+4.6 [CI: -1.3 to 11.0] (r²=0.94). This LC-ESI-MS/MS based method is characterized by simple sample processing and a short run time. The comparison with the current HPLC method is excellent although a significant proportional bias was detected. To conclude, the LC-ESI-MS/MS method is an appropriate method to determine PLP in whole blood.

  13. Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade.

    PubMed

    Kim, Jae-Eung; Zhang, Y-H Percival

    2016-02-01

    Sugar phosphates cannot be produced easily by microbial fermentation because negatively-charged compounds cannot be secreted across intact cell membrane. D-xylulose 5-phosphate (Xu5P), a very expensive sugar phosphate, was synthesized from D-xylose and polyphosphate catalyzed by enzyme cascades in one pot. The synthetic enzymatic pathway comprised of xylose isomerase and xylulokinase was designed to produce Xu5P, along with a third enzyme, polyphosphate kinase, responsible for in site ATP regeneration. Due to the promiscuous activity of the ATP-based xylulokinase from a hyperthermophilic bacterium Thermotoga maritima on polyphosphate, the number of enzymes in the pathway was minimized to two without polyphosphate kinase. The reactions catalyzed by the two-enzyme and three-enzyme pathways were compared for Xu5P production, and the reaction conditions were optimized by examining effects of reaction temperature, enzyme ratio and substrate concentration. The optimized two-enzyme system produced 32 mM Xu5P from 50 mM xylose and polyphosphate after 36 h at 45°C. Biosynthesis of less costly Xu5P from D-xylose and polyphosphate could be highly feasible via this minimized two-enzyme pathway.

  14. Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473.

    PubMed

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

    We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. PMID:27438592

  15. Inhibition of 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase by Lipophilic Phosphonates: SAR, QSAR and Crystallographic Studies

    PubMed Central

    Deng, Lisheng; Diao, Jiasheng; Chen, Pinhong; Pujari, Venugopal; Yao, Yuan; Cheng, Gang; Crick, Dean C.; Venkataram Prasad, B. V.; Song, Yongcheng

    2013-01-01

    1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a novel target for developing new antibacterial (including anti-tuberculosis) and antimalaria drugs. 41 lipophilic phosphonates, representing a new class of DXR inhibitors, were synthesized, among which 5-phenylpyridin-2-ylmethylphosphonic acid possesses the most activity against E. coli DXR (EcDXR) with a Ki of 420 nM. Structure activity relationships (SAR) are discussed, which can be rationalized using our EcDXR:inhibitor structures, and a predictive quantitative SAR (QSAR) model is also developed. Since inhibition studies of DXR from Mycobacterium tuberculosis (MtDXR) have not been well performed, 48 EcDXR inhibitors with a broad chemical diversity were found, however, to generally exhibit considerably reduced activity against MtDXR. The crystal structure of a MtDXR:inhibitor complex reveals the flexible loop containing the residues 198–208 has no strong interactions with the 3,4-dichlorophenyl group of the inhibitor, representing a structural basis for the reduced activity. Overall, these results provide implications in the future design and development of potent DXR inhibitors. PMID:21561155

  16. Thermodynamical characteristics of the reaction of pyridoxal-5'-phosphate with L-amino acids in aqueous buffer solution

    NASA Astrophysics Data System (ADS)

    Barannikov, V. P.; Badelin, V. G.; Venediktov, E. A.; Mezhevoi, I. N.; Guseinov, S. S.

    2011-01-01

    The reaction of pyridoxal-5'-phosphate with L-isomers of alanine, lysine, arginine, aspartic acid, glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calorimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions. The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine. The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the endothermic effect of the reaction increases.

  17. Mechanisms of the beneficial effects of vitamin B6 and pyridoxal 5-phosphate on cardiac performance in ischemic heart disease.

    PubMed

    Dhalla, Naranjan S; Takeda, Satoshi; Elimban, Vijayan

    2013-03-01

    Although vitamin B6 and its metabolite, pyridoxal 5'-phosphate (PLP), have been shown to exert beneficial effects in ischemic heart disease, the mechanisms of their action are not fully understood. Some studies have shown that ventricular arrhythmias and mortality upon the occlusion of coronary artery were attenuated by pretreatment of animals with PLP. Furthermore, ischemia-reperfusion-induced abnormalities in cardiac performance and defects in sarcoplasmic reticular Ca2+-transport activities were decreased by PLP. The increase in cardiac contractile activity of isolated heart by ATP was reduced by PLP, unlike propranolol, whereas that by isoproterenol was not depressed by PLP. ATP-induced increase in [Ca2+]i, unlike KCl-induced increase in [Ca2+]i in cardiomyocytes was depressed by PLP. Both high- and low-affinity sites for ATP binding in sarcolemmal membranes were also decreased by PLP. These observations support the view that PLP may produce cardioprotective effects in ischemic heart disease by attenuating the occurrence of intracellular Ca2+ overload due to the blockade of purinergic receptors.

  18. Fluorescence of the Schiff bases of pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions.

    PubMed

    Cambrón, G; Sevilla, J M; Pineda, T; Blázquez, M

    1996-03-01

    The present study reports on the absorption and emission properties of the Schiff bases formed by pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions. Species protonated at the imine and ring nitrogen are the most fluorescent in both Schiff bases with a quantum yield of 0.02, i.e., 20-fold the value found for species in alkaline solutions. In agreement with other studies, species protonated at the imine nitrogen shows an emission around 500 nm upon excitation at 415 nm. In contrast to previous observations on other PLP Schiff bases, emissions at 560 nm (PL-Ile) and 540 nm (PLP-Ile) are observed upon excitation at 365 and 415 nm, respectively. The emission at 470 nm found in PLP-Ile Schiff base upon excitation at 355 nm is ascribed to a multipolar monoprotonated species. An estimation for the pK a of the imine in the excited state ( ≈ 8.5) for both Schiff bases is also reached. Our results suggest that fast protonation reactions on the excited state are responsible for the observed fluorescence. These effects, in which the hydrogen bond and the phosphate group seem to play a role, could be extended to understanding coenzyme environments in proteins. PMID:24226991

  19. Fluorescence of the Schiff bases of pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions.

    PubMed

    Cambrón, G; Sevilla, J M; Pineda, T; Blázquez, M

    1996-03-01

    The present study reports on the absorption and emission properties of the Schiff bases formed by pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions. Species protonated at the imine and ring nitrogen are the most fluorescent in both Schiff bases with a quantum yield of 0.02, i.e., 20-fold the value found for species in alkaline solutions. In agreement with other studies, species protonated at the imine nitrogen shows an emission around 500 nm upon excitation at 415 nm. In contrast to previous observations on other PLP Schiff bases, emissions at 560 nm (PL-Ile) and 540 nm (PLP-Ile) are observed upon excitation at 365 and 415 nm, respectively. The emission at 470 nm found in PLP-Ile Schiff base upon excitation at 355 nm is ascribed to a multipolar monoprotonated species. An estimation for the pK a of the imine in the excited state ( ≈ 8.5) for both Schiff bases is also reached. Our results suggest that fast protonation reactions on the excited state are responsible for the observed fluorescence. These effects, in which the hydrogen bond and the phosphate group seem to play a role, could be extended to understanding coenzyme environments in proteins.

  20. Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5'-phosphate synthesis and pathogenesis of Magnaporthe oryzae.

    PubMed

    Qi, Zhongqiang; Liu, Muxing; Dong, Yanhan; Yang, Jie; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2016-04-01

    Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5'-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae. PMID:26810198

  1. Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids.

    PubMed

    Schwender, J; Gemünden, C; Lichtenthaler, H K

    2001-02-01

    The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.

  2. Folding pathway of the pyridoxal 5'-phosphate C-S lyase MalY from Escherichia coli.

    PubMed

    Bertoldi, Mariarita; Cellini, Barbara; Laurents, Douglas V; Borri Voltattorni, Carla

    2005-08-01

    MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5'-phosphate) enzyme acting as a beta-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2-4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

  3. Maillard reaction of ribose 5-phosphate generates superoxide and glycation products for bovine heart cytochrome c reduction.

    PubMed

    Gersten, Rebecca A; Gretebeck, Lisa M; Hildick-Smith, Gordon; Sandwick, Roger K

    2010-11-22

    Ribose 5-phosphate (R5P) is a sugar known to undergo the Maillard reaction (glycation) at a rapid rate. In a reaction with the lysines of bovine heart cytochrome c, R5P generates superoxide (O2-) that subsequently reduces ferri-cytochrome c to ferro-cytochrome c. The rate equation for the observed cytochrome c reduction is first order in respect to cytochrome c and half order in respect to R5P. The addition of amines to the cytochrome c-R5P system greatly increases the O2- generation with rates of approximately 1.0 μMmin(-1) being observed with millimolar levels of R5P and amine at 37°C. Pre-incubation of R5P with the amine prior to cytochrome c addition further enhances the rate of cytochrome c reduction approximately twofold for every 30 min of incubation. While clearly accounting for a portion of the reduction of cytochrome c, O2- is not the sole reductant of the system as the use of superoxide dismutase only partially limits cytochrome c reduction, and the contribution of O2- proportionally decreases with longer amine-R5P incubation times. The remainder of the cytochrome c reduction is attributed to either the Amadori product or a cross-linked Schiff base created when a Maillard reaction-derived dicarbonyl compound(s) reacts with the amine. It is believed that these compounds directly transfer electrons to ferri-cytochrome c and subsequently become stable free-radical cations. ATP, a putative regulator of cytochrome c activity, does not inhibit electron transport from O2- or the cross-linked Schiff base but does prevent R5P from reacting with surface lysines to generate superoxide. The spontaneous reaction between R5P and amines could serve as an alternative system for generating O2- in solution. PMID:20933223

  4. Increased D-allose production by the R132E mutant of ribose-5-phosphate isomerase from Clostridium thermocellum.

    PubMed

    Yeom, Soo-Jin; Seo, Eun-Sun; Kim, Yeong-Su; Oh, Deok-Kun

    2011-03-01

    Ribose-5-phosphate isomerase from Clostridium thermocellum converted D-psicose to D-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in D-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for D-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k(cat)/K(m)) of the R132E mutant for D-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of D-psicose to D-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.

  5. Mechanistic studies of 1-aminocyclopropane-1-carboxylate deaminase: characterization of an unusual pyridoxal 5'-phosphate-dependent reaction.

    PubMed

    Thibodeaux, Christopher J; Liu, Hung-Wen

    2011-03-22

    1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give α-ketobutyric acid and ammonia as products. The cleavage of the C(α)-C(β) bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK(a) of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and (13)C KIE studies of the wild type enzyme suggest that the C(α)-C(β) bond cleavage step in the chemical mechanism is at least partially rate-limiting under k(cat)/K(m) conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.

  6. Alkaline phosphatase (tissue-nonspecific isoenzyme) is a phosphoethanolamine and pyridoxal-5'-phosphate ectophosphatase: normal and hypophosphatasia fibroblast study.

    PubMed Central

    Fedde, K N; Whyte, M P

    1990-01-01

    To clarify its physiologic role, alkaline phosphatase (ALP) was examined in normal skin fibroblasts and was shown to be the tissue-nonspecific (TNS) isoenzyme type (as evidenced by heat and inhibition profiles) and to be active toward millimolar concentrations of the putative natural substrates phosphoethanolamine (PEA) and pyridoxal-5'-phosphate (PLP). Fibroblast ALP has a low-affinity activity, with a distinctly alkaline pH optimum (9.3), toward 4-methylumbelliferyl phosphate (4-MUP), PEA, and PLP but a more physiologic pH optimum (8.3) toward physiologic concentrations (micromolar) of PEA and PLP. Normal fibroblast ALP is linked to the outside of the plasma membrane, since in intact cell monolayers (1) dephosphorylation rates of the membrane-impermeable substrates PEA and PLP in the medium at physiologic pH were similar to those observed with disrupted cell monolayers, (2) brief exposure to acidic medium resulted in greater than 90% inactivation of the total ALP activity, and (3) digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) released about 80% of the ALP activity. Hypophosphatasia fibroblasts were markedly deficient (2%-5% control values) in alkaline and physiologic ALP activity when 4-MUP, PLP, and PEA were used as substrate. The majority of the detectable ALP activity, however, appeared to be properly lipid anchored in ecto-orientation. Thus, our findings of genetic deficiency of PEA- and PLP-phosphatase activity in hypophosphatasia fibroblasts, as well as our biochemical findings, indicate that TNS-ALP acts physiologically as a lipid-anchored PEA and PLP ectophosphatase. PMID:2220817

  7. The phosphate of pyridoxal-5'-phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase.

    PubMed

    Phillips, Robert S; Scott, Israel; Paulose, Riya; Patel, Akshay; Barron, Taylor Colt

    2014-02-01

    Kynureninase (L-kynurenine hydrolase, EC 3.7.1.3) catalyzes the hydrolytic cleavage of L-kynurenine to L-alanine and anthranilic acid. The proposed mechanism of the retro-Claisen reaction requires extensive acid/base catalysis. Previous crystal structures showed that Tyr226 in the Pseudomonas fluorescens enzyme (Tyr275 in the human enzyme) hydrogen bonds to the phosphate of the pyridoxal-5'-phosphate (PLP) cofactor. This Tyr residue is strictly conserved in all sequences of kynureninase. The human enzyme complexed with a competitive inhibitor, 3-hydroxyhippuric acid, showed that the ligand carbonyl O is located 3.7 Å from the phenol of Tyr275 (Lima, S., Kumar, S., Gawandi, V., Momany, C. & Phillips, R. S. (2009) J. Med. Chem. 52, 389-396). We prepared a Y226F mutant of P. fluorescens kynureninase to probe the role of this residue in catalysis. The Y226F mutant has approximately 3000-fold lower activity than wild-type, and does not show the pKa values of 6.8 on kcat and 6.5 and 8.8 on k(cat)/K(m) seen for the wild-type enzyme (Koushik, S. V., Moore, J. A. III, Sundararaju, B. & Phillips, R. S. (1998) Biochemistry 37, 1376-1382). Wild-type kynureninase shows a resonance at 4.5 ppm in (31)P-NMR, which is shifted to 5.0, 3.3 and 2.0 ppm when the potent inhibitor 5-bromodihydrokynurenine is added. However, Y226F kynureninase shows resonances at 3.6 and 2.5 ppm, and no change in the peak position is seen when 5-bromodihydrokynurenine is added. Taken together, these results suggest that Tyr226 mediates proton transfer between the substrate and the phosphate, which accelerates formation of external aldimine and gem-diol intermediates. Thus, the phosphate of PLP acts as an acid/base catalyst in the mechanism of kynureninase.

  8. Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-D-ribose 5-phosphate aldolase.

    PubMed

    Li, Jitao; Yang, Jiangang; Men, Yan; Zeng, Yan; Zhu, Yueming; Dong, Caixia; Sun, Yuanxia; Ma, Yanhe

    2015-10-01

    2-Deoxy-D-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERA(K12) (S238D/F200I/ΔY259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards D-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-D-ribose yield from 0.41 mol/mol D-glyceraldehyde to 0.81 mol/mol D-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-D-robose (DR), with a yield of 0.71 mol/mol D-glyceraldehyde and average productivity of 0.13 mol/L·h (17.94 g/L·h). These results demonstrate the potential for large-scale production of 2-deoxy-D-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-D-altrose from D-erythrose, as well as 2-deoxy-L-xylose and 2-deoxy-L-ribose from L-glyceraldehyde.

  9. Structural basis of the tight binding of pyridoxal 5'-phosphate to a low molecular weight protein tyrosine phosphatase.

    PubMed

    Zhou, M; Van Etten, R L

    1999-03-01

    Pyridoxal 5'-phosphate (PLP) binds tightly to bovine low Mr protein tyrosine phosphatase (BPTP), but it is a very poor substrate for the enzyme. The structural basis of this tight binding of PLP is examined here by a variety of methods. Binding constants of a number of PLP analogues were measured with wild-type BPTP, and PLP binding constants of some site-specific mutants of BPTP were determined at pH 5.0 through the use of several independent methods. The tight binding of PLP (Ki = 7.6 microM) causes a downfield shift of the His-72 Cepsilon1H resonance in the 1H NMR spectrum of the protein, consistent with a structural alteration in the phosphate binding loop transmitted through a complex hydrogen bond network that exists between His-72 and Asn-15, which is a residue in the phosphate binding loop. 1H NMR spectroscopy with an MLEV-17 spectral editing scheme was used to monitor the aldehyde resonance of PLP during titration of a catalytically inactive C12A mutant of BPTP. The aldehydic proton resonance of PLP shifted from 10.43 to 10.26 ppm upon complex formation with the C12A mutant. This resonance occurs far from the region where a hemithioacetal hydrogen would be expected to appear, consistent with the conclusion that the Cys-17 side chain of BPTP does not add to the aldehyde group of PLP. UV-visible spectrophotometric titration also supported this conclusion. The binding constant of PLP to a C17A mutant was similar to that exhibited with wild-type protein. These results show that Cys-17 makes virtually no contribution to the tight binding of PLP by BPTP, in contrast to a published report that it is "essential" for binding PLP. On the other hand, Asp-129 of BPTP was found to be very important for binding PLP. It is concluded that Asp-129 binds to the pyridinium nitrogen of PLP and that this renders Asp-129 effectively unavailable to serve its essential catalytic role as a general acid. The interactions described here should be useful in the design of specific

  10. [Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum].

    PubMed

    Zhang, Chun-Rong; Yang, Quan; Chen, Hu-Biao; Pang, Yu-Xin; Tang, Xiao-Min; Cheng, Xuan-Xuan; Wu, Wen-Ya; Chen, Shi-Min

    2012-11-01

    The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.

  11. Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi

    PubMed Central

    da Silva, Tiago Fernandes; Palhano, Fernando L.

    2015-01-01

    Pyridoxinamine 5'-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases. PMID:26327315

  12. Effects of 2'-deoxycoformycin, 9-beta-D-arabinofuranosyladenine 5'-phosphate, and 1-beta-D-arabinofuranosylcytosine triple combination therapy on intracerebral leukemia 1210.

    PubMed

    Caron, N; Lee, S H; Kimball, A P

    1977-09-01

    The triple combination of 2'-deoxycoformycin (2'-dCF), 9-beta-D-arabinofuranosyladenine 5'-phosphate, and 9-beta-D-arabinofuranosylcytosine was found to be very effective in the therapy of C57BL X DBA/2 F1 mice with intracerebral L1210. At the dosages and dosage scheduling used, the double combination of 2'-dCF and 9-beta-D-arabinofuranosyladenine 5'-phosphate gave minimal but significant increases in life-span. When 9-beta-D-arabinofuranosylcytosine was given at suboptimal dosage to mice with intracerebral L1210, the host toxicity caused by 2'-dCF and 9-beta-D-arabinofuranosyladenine 5'-phosphate in combination was decreased by a factor of 2, allowing a more prolonged therapy. "Cures" were obtained with the triple combination at dosages of 9-beta-D-arabinofuranosylcytosine that did not "cure". The supernatant adenosine deaminase from C57BL X DBA/2 F1 mouse brains was purified and the Ki for 2'-dCF using 9-beta-D-arabinofuranosyladenine as substrate was determined to be not more than 2 X 10(-11) M.

  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. Structural Insights into the Mechanism of the PLP Synthase Holoenzyme from Thermotoga maritima†,‡

    PubMed Central

    Zein, Fairuz; Zhang, Yan; Kang, You-Na; Burns, Kristin; Begley, Tadhg P.; Ealick, Steven E.

    2008-01-01

    Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino-sugars. The PLP synthase holoenzyme consists of two subunits: YaaD catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate and ammonia and YaaE catalyzes the production of ammonia from glutamine. Here we describe the structure of the PLP synthase complex (YaaD-YaaE) from Thermotoga maritima at 2.9 Å resolution. This complex consists of a core of 12 YaaD monomers with 12 noninteracting YaaE monomers attached to the core. Compared to the previously published structure of PdxS (a YaaD ortholog in Geobacillus stearothermophilus), the N-terminus (1–18), which includes helix α0, the β2-α2 loop(46–56), which includes new helix α2a, and the C-terminus (270–280) of YaaD, are ordered in the complex but disordered in PdxS. A ribulose 5-phosphate is bound to YaaD via an imine with Lys82. Previous studies have demonstrated a similar imine at Lys149 and not at Lys81 (equivalent to Lys150 and 82 in T. maritima) for the Bacillus subtilis enzyme suggesting the possibility that two separate sites on YaaD are involved in PLP formation. A phosphate from the crystallization solution is found bound to YaaD and also serves as a marker for a possible second active site. An ammonia channel that connects the active site of YaaE with the ribulose 5-phosphate binding site was identified. This channel is similar to one found in imidazole glycerol phosphate synthase; however, when the β-barrels of the two complexes are superimposed, the glutaminase domains are rotated by about 180° with respect to each other. PMID:17144654

  16. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    PubMed Central

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  17. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

    PubMed

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  18. [Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts].

    PubMed

    Logvinenko, E M; Shavlovskiĭ, G M; Zakal'skiĭ, A E; Kontorovskaia, N Iu

    1989-01-01

    2,5-Diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of Pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (NH4)2SO4 at 45-75% of saturation and chromatography on blue sepharose CL-6B. The use of gel filtration through Sephadex G-150 and chromatography on DEAE-cellulose proved to be less effective for the enzyme purification. It has been established that it is 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5-phosphate but not its dephosphorylated form that is the substrate of the given reductase; Km is equal to 7.10(-5) M. The reaction proceeds in the presence of NADPH or NADH. The enzyme affinity to NADPH (Km = 4.7.10(-5) M) is approximately one order higher than that to NADPH (Km = 5.5.10(-4) M). The enzyme manifests the optimum of action at pH 7.2 and the temperature of 37 degrees C; the molecular weight is 140 kD. EDTA as well as flavins in the concentration of 1.10(-3) M exert no effect on the reductase activity. The enzyme is labile at 4 degrees C and is inactivated in the frozen state at -15 degrees C. The 2.5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been also revealed in Torulopsis candida, Debaryomyces klöckeri, Schwanniomyces occidentalis, Eremothecium ashbyii (flavinogenic species) and Candida utilis. Aspergillus nidulans, Neurospora crassa (nonflavinogenic species). The synthesis of this enzyme contrary to other enzymes of the riboflavin biosynthesis is not regulated in flavinogenic yeast by iron ions. PMID:2511652

  19. [Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts].

    PubMed

    Logvinenko, E M; Shavlovskiĭ, G M; Zakal'skiĭ, A E; Kontorovskaia, N Iu

    1989-01-01

    2,5-Diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of Pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (NH4)2SO4 at 45-75% of saturation and chromatography on blue sepharose CL-6B. The use of gel filtration through Sephadex G-150 and chromatography on DEAE-cellulose proved to be less effective for the enzyme purification. It has been established that it is 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5-phosphate but not its dephosphorylated form that is the substrate of the given reductase; Km is equal to 7.10(-5) M. The reaction proceeds in the presence of NADPH or NADH. The enzyme affinity to NADPH (Km = 4.7.10(-5) M) is approximately one order higher than that to NADPH (Km = 5.5.10(-4) M). The enzyme manifests the optimum of action at pH 7.2 and the temperature of 37 degrees C; the molecular weight is 140 kD. EDTA as well as flavins in the concentration of 1.10(-3) M exert no effect on the reductase activity. The enzyme is labile at 4 degrees C and is inactivated in the frozen state at -15 degrees C. The 2.5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been also revealed in Torulopsis candida, Debaryomyces klöckeri, Schwanniomyces occidentalis, Eremothecium ashbyii (flavinogenic species) and Candida utilis. Aspergillus nidulans, Neurospora crassa (nonflavinogenic species). The synthesis of this enzyme contrary to other enzymes of the riboflavin biosynthesis is not regulated in flavinogenic yeast by iron ions.

  20. Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5'-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

    PubMed Central

    Donoso, J; Muñoz, F; García Del Vado, A; Echevarría, G; García Blanco, F

    1986-01-01

    Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate. PMID:3099764

  1. The 2.2 A resolution structure of RpiB/AlsB from Escherichia coli illustrates a new approach to the ribose-5-phosphate isomerase reaction.

    PubMed

    Zhang, Rong-Guang; Andersson, C Evalena; Skarina, Tatiana; Evdokimova, Elena; Edwards, Aled M; Joachimiak, Andrzej; Savchenko, Alexei; Mowbray, Sherry L

    2003-10-01

    Ribose-5-phosphate isomerases (EC 5.3.1.6) interconvert ribose 5-phosphate and ribulose 5-phosphate. This reaction permits the synthesis of ribose from other sugars, as well as the recycling of sugars from nucleotide breakdown. Two unrelated types of enzyme can catalyze the reaction. The most common, RpiA, is present in almost all organisms (including Escherichia coli), and is highly conserved. The second type, RpiB, is present in some bacterial and eukaryotic species and is well conserved. In E.coli, RpiB is sometimes referred to as AlsB, because it can take part in the metabolism of the rare sugar, allose, as well as the much more common ribose sugars. We report here the structure of RpiB/AlsB from E.coli, solved by multi-wavelength anomalous diffraction (MAD) phasing, and refined to 2.2A resolution. RpiB is the first structure to be solved from pfam02502 (the RpiB/LacAB family). It exhibits a Rossmann-type alphabetaalpha-sandwich fold that is common to many nucleotide-binding proteins, as well as other proteins with different functions. This structure is quite distinct from that of the previously solved RpiA; although both are, to some extent, based on the Rossmann fold, their tertiary and quaternary structures are very different. The four molecules in the RpiB asymmetric unit represent a dimer of dimers. Active-site residues were identified at the interface between the subunits, such that each active site has contributions from both subunits. Kinetic studies indicate that RpiB is nearly as efficient as RpiA, despite its completely different catalytic machinery. The sequence and structural results further suggest that the two homologous components of LacAB (galactose-6-phosphate isomerase) will compose a bi-functional enzyme; the second activity is unknown.

  2. The Arabidopsis homolog of trithorax, ATX1, binds phosphatidylinositol 5-phosphate, and the two regulate a common set of target genes.

    SciTech Connect

    Alvarez-Venegas,R.; Sadder, M.; Hlavacka, A.; Baluska, F.; Xia, Y.; Firsov, A.; Sarath, G.; Moriyama, H.; Dubrovsky, J.; Avramova, Z.

    2006-01-01

    The Arabidopsis homolog of trithorax, ATX1, regulates numerous functions in Arabidopsis beyond the homeotic genes. Here, we identified genome-wide targets of ATX1 and showed that ATX1 is a receptor for a lipid messenger, phosphatidylinositol 5-phosphate, PI5P. PI5P negatively affects ATX1 activity, suggesting a regulatory pathway connecting lipid-signaling with nuclear functions. We propose a model to illustrate how plants may respond to stimuli (external or internal) that elevate cellular PI5P levels by altering expression of ATX1-controlled genes.

  3. 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.

  4. A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas

    PubMed Central

    2012-01-01

    Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. Methods We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. Results and conclusions Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples. PMID:22995035

  5. Characterization of a human X-linked gene from the DXS732E locus in the candidate region for the anhidrotic ectodermal dysplasia (EDA) gene (Xq13.1)

    SciTech Connect

    Gault, J.; Zonana, J.; Zeltinger, J.

    1994-09-01

    A conserved mouse genomic clone was used to identify a homologous human genomic clone (the DXS732E locus), which was subsequently employed to isolate cDNAs from a human fetal brain library. Nine unique overlapping cDNAs were isolated, and sequences analysis of 3.9 kb identified a putative 1 kb ORF. GRAIL analysis of the sequence supported the hypothesis that the putative ORF was coding sequence, and Prosite analysis of the putative ORF identified potential glycosylation and phosphorylation sites. The 5{prime} end of the gene maps within a CpG island, and comparison of cDNA sequences indicate the gene is alternatively spliced at its 3{prime} end. Northern analysis and RT-PCR indicate that two different sized messages appear to be expressed with the gene expressed in human fetal kidney, intestine, brain, and muscle. The gene is expressed in 77 day human skin, a time when hair follicle formation occurs. Anhidrotic ectodermal dysplasia (EDA) results in the abnormal morphogenesis of hair, teeth and eccrine sweat glands. A positional cloning strategy towards cloning the EDA gene had been used, and deletion and X-autosome translocation patients have been useful in further delimiting the EDA region. The present gene at the DXS732E locus is partially deleted in one EDA patient who does not have other apparent abnormalities. No rearrangements of the gene have been detected in two female X-autosome translocation EDA patients, nor in four additional male patients with submicroscopic molecular deletions.

  6. Conserved YjgF protein family deaminates reactive enamine/imine intermediates of pyridoxal 5'-phosphate (PLP)-dependent enzyme reactions.

    PubMed

    Lambrecht, Jennifer A; Flynn, Jeffrey M; Downs, Diana M

    2012-01-27

    The YjgF/YER057c/UK114 family of proteins is conserved in all domains of life, suggesting that the role of these proteins arose early and was maintained throughout evolution. Metabolic consequences of lacking this protein in Salmonella enterica and other organisms have been described, but the biochemical function of YjgF remained unknown. This work provides the first description of a conserved biochemical activity for the YjgF protein family. Our data support the conclusion that YjgF proteins have enamine/imine deaminase activity and accelerate the release of ammonia from reactive enamine/imine intermediates of the pyridoxal 5'-phosphate-dependent threonine dehydratase (IlvA). Results from structure-guided mutagenesis experiments suggest that YjgF lacks a catalytic residue and that it facilitates ammonia release by positioning a critical water molecule in the active site. YjgF is renamed RidA (reactive intermediate/imine deaminase A) to reflect the conserved activity of the protein family described here. This study, combined with previous physiological studies on yjgF mutants, suggests that intermediates of pyridoxal 5'-phosphate-mediated reactions may have metabolic consequences in vivo that were previously unappreciated. The conservation of the RidA/YjgF family suggests that reactive enamine/imine metabolites are of concern to all organisms.

  7. Structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin

    SciTech Connect

    Yajima, Shunsuke Hara, Kodai; Iino, Daisuke; Sasaki, Yasuyuki; Kuzuyama, Tomohisa; Ohsawa, Kanju; Seto, Haruo

    2007-06-01

    The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg{sup 2+}, NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR. The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg{sup 2+}, NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg{sup 2+} was shifted by NADPH in the active site.

  8. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    SciTech Connect

    Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W.

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  9. Expression of nitric oxide synthase in rat glomerular mesangial cells mediated by cyclic AMP.

    PubMed Central

    Mühl, H.; Kunz, D.; Pfeilschifter, J.

    1994-01-01

    1. Treatment of rat mesangial cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) has been shown to induce a macrophage-type of nitric oxide (NO) synthase. Here we report that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is another mediator that triggers induction of NO synthase in mesangial cells. 2. Incubation of mesangial cells with the beta-adrenoceptor agonist, salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cyclic AMP concentration, increased nitrite formation in a dose-dependent manner. Likewise, the addition of the membrane-permeable cyclic AMP analogue, N6, 0-2'-dibutyryladenosine 3',5'-phosphate (Bt2 cyclic AMP) or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine enhanced NO synthase activity in a dose-dependent manner. 3. There was a lag period of about 8 h before a significantly enhanced secretion of nitrite could be detected upon exposure of cells to forskolin and for maximal stimulation, forskolin had to be present during the whole incubation period. 4. Treatment of mesangial cells with actinomycin D, cycloheximide or dexamethasone completely suppressed forskolin-stimulated NO-synthase activity, thus demonstrating that transcription and protein synthesis are necessary for nitrite formation. 5. Bt2 cyclic AMP, the most potent inducer of nitrite production, increased NO synthase mRNA levels in mesangial cells in a time- and dose-dependent fashion. Dexamethasone completely inhibited the increase of NO synthase mRNA in response to Bt2 cyclic AMP. 6. Combination of Bt2 cyclic AMP and IL-1 beta or TNF alpha revealed a strong synergy in terms of nitrite formation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 7 Figure 8 Figure 9 PMID:7518300

  10. 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.

  11. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    SciTech Connect

    Akana,J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

    2006-01-01

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common ({beta}/{alpha}){sub 8}-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn{sup 2+} which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn{sup 2+} and inactive apoenzyme cannot be prepared, the affinity for Zn{sup 2+} is decreased by alanine substitutions for the two histidine residues that coordinate the Zn{sup 2+} ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn{sup 2+}. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn{sup 2+} that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn{sup 2+} and participate as acid/base catalysts are not

  12. Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

    PubMed

    Capriles, Priscila V S Z; Baptista, Luiz Phillippe R; Guedes, Isabella A; Guimarães, Ana Carolina R; Custódio, Fabio L; Alves-Ferreira, Marcelo; Dardenne, Laurent E

    2015-02-01

    Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog.

  13. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

    PubMed

    Saladié, Montserrat; Wright, Louwrance P; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A

    2014-09-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties. PMID

  14. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps

    PubMed Central

    Saladié, Montserrat; Wright, Louwrance P.; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A.

    2014-01-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties. PMID

  15. The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5'-phosphate from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate and AMP.

    PubMed

    Krall, Lilian; Raschke, Maja; Zenk, Meinhart H; Baron, Christian

    2002-09-11

    The plant pathogen Agrobacterium tumefaciens produces cytokinins upon induction of the virulence genes by secondary metabolites from wounded plants, and these hormones are believed to stimulate the infection process. To study the biosynthetic pathway, the tzs gene, encoding the Tzs (trans-zeatin-synthesizing) protein from A. tumefaciens, was cloned and the protein was overproduced and purified. Analysis of its reactivity with radioactively labeled substrate demonstrated conversion of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate, a product of the deoxyxylulose phosphate pathway, with AMP to zeatin riboside 5'-phosphate. This suggests that A. tumefaciens uses an alternative pathway of cytokinin biosynthesis, which had previously been hypothesized to operate in plants. PMID:12220681

  16. K30, H150, and H168 are essential residues for coordinating pyridoxal 5'-phosphate of O-acetylserine sulfhydrylase from Acidithiobacillus ferrooxidans.

    PubMed

    Zheng, Chunli; Nie, Li; Qian, Lin; Wang, Zhilou; Liu, Guizhen; Liu, Jianshe

    2010-06-01

    O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV-vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5'-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization. PMID:20033172

  17. Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate.

    PubMed

    Ravi, G; Venkatesh, Yeldur P

    2014-11-01

    D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI-TOF analyses, an increase in apparent molecular weight (SDS-PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P-BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P-BSA antiserum subjected to caprylic acid precipitation followed by hapten-affinity chromatography; its affinity constant is 7.1 × 10(8) M(-1). Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P-specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P-protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P-specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide. PMID:25108762

  18. Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase).

    PubMed Central

    Miller, A D; Packman, L C; Hart, G J; Alefounder, P R; Abell, C; Battersby, A R

    1989-01-01

    A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site. PMID:2510713

  19. Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

    SciTech Connect

    Dotson, G.D.; Woodard, R.W.

    1994-12-01

    The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3{sup 2}H)PEP, (2-{sup 13}C)PEP, and (2-{sup 13}C,{sup 18}O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our {sup 1}H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3-{sup 2}H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3-{sup 2}H)PEP gave predominantly (3S)-(3{sup 2}H)KDO 8-P and (E)-(3-{sup 2}H)PEP gave predominantly (3R)-(3{sup 2}H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2-{sup 13}C, {sup 18}O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both {sup 13}C- and {sup 31}P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the {sup 18}O.

  20. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    SciTech Connect

    Kouchi, Zen; Fujiwara, Yuki; Yamaguchi, Hideki; Nakamura, Yoshikazu; Fukami, Kiyoko

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

  1. Thermodynamic and microscopic equilibrium constants of molecular species formed from pyridoxal 5'-phosphate and 2-amino-3-phosphonopropionic acid in aqueous and D/sub 2/O solution

    SciTech Connect

    Szpoganicz, B.; Martell, A.E.

    1984-09-19

    Schiff base formation between pyridoxal 5'-phosphate (PLP) and 2-amino-3-phosphonopropionic acid (APP) has been investigated by measurement of the corresponding NMR and electronic absorption spectra. A value of 0.26 was found for the formation constant of the completely deprotonated Schiff base species, and is much smaller than the values reported for pyridoxal-..beta..-chloroalanine and pyridoxal-O-phosphoserine. The protonation constants for the aldehyde and hydrate forms of PLP were determined in D/sub 2/O by measurement of the variation of chemical shifts with pD (pH in D/sub 2/O). The hydration constants of PLP were determined in a pD range 2-12, and species distributions were calculated. The protonation constants of the APP-PLP Schiff base determined by NMR in D/sub 2/O were found to have the log values 12.54, 8.10, 6.70, and 5.95, and the species distributions were calculated for a range of pD values. Evidence is reported for hydrogen bonding involving the phosphate and phosphonate groups of the diprotonated Schiff base. The cis and trans forms of the Schiff bases were distinguished with the aid of the nuclear Overhauser effect. 43 references, 9 figures, 3 tables.

  2. Efficient production of a thermophilic 2-deoxyribose-5-phosphate aldolase in glucose-limited fed-batch cultivations of Escherichia coli by continuous lactose induction strategy.

    PubMed

    Pei, Xiao-Lin; Wang, Qiu-Yan; Li, Cheng-Lu; Qiu, Xiao-Feng; Xie, Kai-Lin; Huang, Li-Feng; Wang, An-Ming; Zeng, Zhao-Wu; Xie, Tian

    2011-09-01

    The production of a thermophilic 2-deoxyribose-5-phosphate aldolases (DERA) in Escherichia coli BL21 under continuous lactose induction strategy was investigated. The process was combined with the exponential feeding method, controlling the feeding rate to maintain the specific growth rate at 0.15 h(-1). The results indicate that the lactose concentration in the feed medium affected directly the expression of the target protein. The use of 50 g/L in the feed medium resulted in the biomass concentration of 39.3 g DCW/L, and an expression level of above 30%, and the maximum final DERA concentration of 16,200 U/L. Furthermore, the acetate concentration remained at a low level in the fed-batch phase, less than 0.5 g/L. In conclusion, combining glucose feeding with lactose induction is a more powerful way to achieve high cell density cultures and to efficiently produce the thermophilic DERA. The results also indicate the potential industrial utility in the scale production of other recombinant proteins. PMID:21509600

  3. Cloning, Characterization, and Immunolocalization of a Mycorrhiza-Inducible 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase in Arbuscule-Containing Cells of Maize1

    PubMed Central

    Hans, Joachim; Hause, Bettina; Strack, Dieter; Walter, Michael H.

    2004-01-01

    Colonization of plant roots by symbiotic arbuscular mycorrhizal fungi frequently leads to the accumulation of several apocarotenoids. The corresponding carotenoid precursors originate from the plastidial 2-C-methyl-d-erythritol 4-phosphate pathway. We have cloned and characterized 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzing the first committed step of the pathway, from maize (Zea mays). Functional identification was accomplished by heterologous expression of sequences coding for the mature protein in Escherichia coli. DXR is up-regulated in maize roots during mycorrhization as shown at transcript and protein levels, but is also abundant in leaves and young seedlings. Inspection of sequenced genomes and expressed sequence tag (EST) databases argue for a single-copy DXR gene. Immunolocalization studies in mycorrhizal roots using affinity-purified antibodies revealed a DXR localization in plastids around the main symbiotic structures, the arbuscules. DXR protein accumulation is tightly correlated with arbuscule development. The highest level of DXR protein is reached around maturity and initial senescence of these structures. We further demonstrate the formation of a DXR-containing plastidial network around arbuscules, which is highly interconnected in the mature, functional state of the arbuscules. Our findings imply a functional role of a still unknown nature for the apocarotenoids or their respective carotenoid precursors in the arbuscular life cycle. PMID:14764905

  4. Quantum mechanics/molecular mechanics studies on the mechanism of action of cofactor pyridoxal 5'-phosphate in ornithine 4,5-aminomutase.

    PubMed

    Pang, Jiayun; Scrutton, Nigel S; Sutcliffe, Michael J

    2014-09-01

    A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions.

  5. Comparative protein modeling of 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme from Plasmodium falciparum: a potential target for antimalarial drug discovery.

    PubMed

    Singh, Nidhi; Chevé, Gwénaël; Avery, Mitchell A; McCurdy, Christopher R

    2006-01-01

    Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Pf-DXR) is a potential target for antimalarial chemotherapy. The three-dimensional model (3D) of this enzyme was determined by means of comparative modeling through multiple alignment followed by intensive optimization, minimization, and validation. The resulting model demonstrates a reasonable topology as gauged from the Ramachandran plot and acceptable three-dimensional structure compatibility as assessed by the Profiles-3D score. The modeled monomeric subunit consists of three domains: (1) N-terminal NADPH binding domain, (2) connective or linker domain (with most of the active site residues located in this domain), and (3) a C-terminal domain. This structure proved to be consistent with known DXR crystal structures from other species. The predicted active site compared favorably with those of the templates and appears to have an active site with a highly conserved architecture. Additionally, the model explains several site-directed mutagenesis data. Besides using several protein structure-checking programs to validate the model, a set of known inhibitors of DXR were also docked into the active site of the modeled Pf-DXR. The docked scores correlated reasonably well with experimental pIC50 values with a regression coefficient (R2) equal to 0.84. Results of the current study should prove useful in the early design and development of inhibitors by either de novo drug design or virtual screening of large small-molecule databases leading to development of new antimalarial agents. PMID:16711755

  6. Crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase from the hyperthermophile Thermotoga maritima for insights into the coordination of conformational changes and an inhibitor binding.

    PubMed

    Takenoya, Mihoko; Ohtaki, Akashi; Noguchi, Keiichi; Endo, Kiwamu; Sasaki, Yasuyuki; Ohsawa, Kanju; Yajima, Shunsuke; Yohda, Masafumi

    2010-06-01

    Isopentenyl diphosphate is a precursor of various isoprenoids and is produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids of plants, protozoa and many eubacteria. A key enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be the target of fosmidomycin, which works as an antimalarial, antibacterial and herbicidal compound. In this paper, we report studies of kinetics and the crystal structures of the thermostable DXR from the hyperthermophile Thermotoga maritima. Unlike the mesophilic DXRs, Thermotoga DXR (tDXR) showed activity only with Mg(2+) at its growth temperature. We solved the crystal structures of tDXR with and without fosmidomycin. The structure without fosmidomycin but unexpectedly bound with 2-methyl-2,4-pentanediol (MPD), revealing a new extra space available for potential drug design. This structure adopted the closed form by rigid domain rotation but without the flexible loop over the active site, which was considered as a novel conformation. Further, the conserved Asp residue responsible for cation binding seemed to play an important role in adjusting the position of fosmidomycin. Taken together, our kinetic and the crystal structures illustrate the binding mode of fosmidomycin that leads to its slow, tight binding according to the conformational changes of DXR.

  7. Cloning, characterization, and immunolocalization of a mycorrhiza-inducible 1-deoxy-d-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize.

    PubMed

    Hans, Joachim; Hause, Bettina; Strack, Dieter; Walter, Michael H

    2004-02-01

    Colonization of plant roots by symbiotic arbuscular mycorrhizal fungi frequently leads to the accumulation of several apocarotenoids. The corresponding carotenoid precursors originate from the plastidial 2-C-methyl-d-erythritol 4-phosphate pathway. We have cloned and characterized 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzing the first committed step of the pathway, from maize (Zea mays). Functional identification was accomplished by heterologous expression of sequences coding for the mature protein in Escherichia coli. DXR is up-regulated in maize roots during mycorrhization as shown at transcript and protein levels, but is also abundant in leaves and young seedlings. Inspection of sequenced genomes and expressed sequence tag (EST) databases argue for a single-copy DXR gene. Immunolocalization studies in mycorrhizal roots using affinity-purified antibodies revealed a DXR localization in plastids around the main symbiotic structures, the arbuscules. DXR protein accumulation is tightly correlated with arbuscule development. The highest level of DXR protein is reached around maturity and initial senescence of these structures. We further demonstrate the formation of a DXR-containing plastidial network around arbuscules, which is highly interconnected in the mature, functional state of the arbuscules. Our findings imply a functional role of a still unknown nature for the apocarotenoids or their respective carotenoid precursors in the arbuscular life cycle.

  8. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives. PMID:27439219

  9. Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability

    PubMed Central

    Kaur, Preet Kamal; Tripathi, Neha; Desale, Jayesh; Neelagiri, Soumya; Yadav, Shailendra; Bharatam, Prasad V.; Singh, Sushma

    2016-01-01

    Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB. PMID:26953696

  10. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives.

  11. Structure of L-xylulose-5-Phosphate 3-epimerase (UlaE) from the anaerobic L-ascorbate utilization pathway of Escherichia coli: identification of a novel phosphate binding motif within a TIM barrel fold.

    PubMed

    Shi, Rong; Pineda, Marco; Ajamian, Eunice; Cui, Qizhi; Matte, Allan; Cygler, Miroslaw

    2008-12-01

    Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of l-ascorbate under anaerobic conditions. UlaD catalyzes a beta-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel beta-strands. The enzyme binds Zn(2+), which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the beta1/alpha1 loop and alpha3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands beta7 and beta8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

  12. Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

    SciTech Connect

    Shi, Rong; Pineda, Marco; Ajamian, Eunice; Cui, Qizhi; Matte, Allan; Cygler, Miroslaw

    2009-01-15

    Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+}, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

  13. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

    PubMed

    Kai, Guoyin; Xu, Hui; Zhou, Congcong; Liao, Pan; Xiao, Jianbo; Luo, Xiuqin; You, Lijia; Zhang, Lin

    2011-05-01

    Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.

  14. NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

    PubMed

    Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M; Compton, Shara; Fogle, Emily; Toney, Michael D; Shenderovich, Ilya; Denisov, Gleb S; Limbach, Hans-Heinrich

    2013-12-01

    Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism. PMID:24147985

  15. Competitive inhibitors of type B ribose 5-phosphate isomerases: design, synthesis and kinetic evaluation of new D-allose and D-allulose 6-phosphate derivatives.

    PubMed

    Mariano, Sandrine; Roos, Annette K; Mowbray, Sherry L; Salmon, Laurent

    2009-05-12

    This study reports syntheses of d-allose 6-phosphate (All6P), D-allulose (or D-psicose) 6-phosphate (Allu6P), and seven D-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichiacoli (EcRpiB). 5-Phospho-D-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho-D-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho-D-ribonohydroxamic acid and N-(5-phospho-D-ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho-D-ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho-D-ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K(i) value of 9 microM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.

  16. Enhancement of corneal permeation of riboflavin-5'-phosphate through vitamin E TPGS: a promising approach in corneal trans-epithelial cross linking treatment.

    PubMed

    Ostacolo, Carmine; Caruso, Ciro; Tronino, Diana; Troisi, Salvatore; Laneri, Sonia; Pacente, Luigi; Del Prete, Antonio; Sacchi, Antonia

    2013-01-20

    Corneal accumulation of riboflavin-5'-phosphate (riboflavin) is an essential step in the so called corneal cross-linking (CXL), an elective therapy for the treatment of progressive keratoconus, corneal ectasia and irregular astigmatism. CXL is usually performed after surgical debridement of corneal epithelium, since it impedes the stromal penetration of riboflavin in a relatively short time. d-Alpha-tocopheryl poly(ethylene glycol) 1000 succinate (VE-TPGS) is an effective permeation enhancer used to increase adsorption of drugs trough different biological barriers. Moreover, belonging to the group of tocopherol pro-drugs, VE-TPGS exerts a protective effect on biological membrane against free-radical damage. The aim of this work is the evaluation of VE-TPGS effects on riboflavin corneal permeability, and the assessment of its protective effect against free-radicals generated during CXL procedures. Different solutions containing riboflavin (0.125% w/w), dextran (20.0% w/w) and increasing concentration of VE-TPGS were tested. Corneal permeation was evaluated in vitro by the use of modified Franz-cell type diffusion cells and freshly excised porcine corneas as barrier. The effect of VE-TPGS on riboflavin corneal penetration was compared with a standard commercial solution of riboflavin in dextran at different times. Accumulation experiments were conducted both on epithelized and non-epithelized corneas. Moreover, epithelized porcine corneas, treated with the tested solutions, were subjected to an in vitro CXL procedure versus non-epithelized corneas, treated with a commercial solution of riboflavin. Differences were measured by means of corneal rigidity using Young's modulus. The photo-protective effect of tested solutions on corneal epithelium was, finally, evaluated. CXL treatment was applied, in vitro, on human explanted corneas and resulting morphology of corneal epithelium was investigated by scanning electron microscopy. PMID:23046664

  17. Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

    PubMed

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2014-11-01

    There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

  18. Bimetallic magnetic nanoparticle as a new platform for fabrication of pyridoxine and pyridoxal-5'-phosphate imprinted polymer modified high throughput electrochemical sensor.

    PubMed

    Patra, Santanu; Roy, Ekta; Das, Ranajit; Karfa, Paramita; Kumar, Sunil; Madhuri, Rashmi; Sharma, Prashant K

    2015-11-15

    The present work describes the fabrication of a selective and sensitive molecularly imprinted polymer (MIP)-based electrochemical sensor using a combination of surface imprinting and nanotechnology. The fabricated sensor was used for the detection of two major components of vitamin B6 i.e. pyridoxine (Py) and pyridoxal-5'-phosphate (PLP) using the same MIP format. Herein, acrylic acid modified zero valent iron nanoparticles were combined with the copper nanoparticle, resulting in vinyl groups modified bimetallic Fe/Cu magnetic nanoparticles (BMNPs). These BMNPs have high surface to volume ratios, higher electro-catalytic activity, and are therefore, a suitable platform to synthesize specific MIP cavities for Py and PLP. Herein, two different MIP formats (for Py and PLP) were synthesized on the surface of vinyl silane modified pencil graphite electrodes by activator regenerated by an electron transfer-atom transfer radical polymerization (ARGET-ATRP) method. The sensor shows a good analytical performance for the detection of Py and PLP by a square wave stripping voltammetric technique (SWSV). The limit of detection (LOD) was calculated to be 0.040 µg L(-1) and 0.043 µg L(-1) for Py and PLP, respectively, at signal to noise ratio of 3. The sensors are highly selective for the templates and can detect them from multivitamin tablets, corn flakes, energy drinks, cerebrospinal fluid (CSF) and blood samples (serum, plasma and whole blood) without any interfering effect, suggesting the clinical applicability of the fabricated sensor. The sensor can also be used as better alternative to the commercially available ELISA kits which are rather complex, less sensitive and difficult to handle.

  19. Alteration of the flexible loop in 1-deoxy-D-xylulose-5-phosphate reductoisomerase boosts enthalpy-driven inhibition by fosmidomycin.

    PubMed

    Kholodar, Svetlana A; Tombline, Gregory; Liu, Juan; Tan, Zhesen; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

    2014-06-01

    1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy-enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin's phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.

  20. Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose or by use of Sepharose-bound pyridoxal 5'-phosphate.

    PubMed

    Fukui, S; Ikeda, S; Fujimura, M; Yamada, H; Kumagai, H

    1975-02-01

    Tryptophanase from Escherichia coli B/qt 7-A and tyrosine phenol-lyase (beta-tyrosinase) from Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5'-phosphate previously bound to Sepharose. The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal-P bound on Sepharose at the 6-position through a diazo linkage. This immobilization procedure involves the formation to Schiff base linkage between 4-formyl group of Sepharose-bound pyridoxal-P and the epsilon-amino group of the lysine residue at the active center of one subunit of tetrameric apo-tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4. In the case of beta-tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase. Direct coupling of the apoenzyme to CNBr-activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results. In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization. When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal-P, immobilized holo-tryptophanase of holo-beta-tyrosinase gradually lost its original activity; however, supplement of pyridoxal-P to the reaction system restored its initial activity. From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and beta-tyrosinase were calculated. Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side. Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guinidine-HCl than their free counterpart. Furthermore, the reactivity of sulfhydryl group of beta-tyrosinase, in connection with its coenzyme-binding property, was conveniently studied by use of the immobilized enzyme.

  1. Disruption of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene results in albino, dwarf and defects in trichome initiation and stomata closure in Arabidopsis.

    PubMed

    Xing, Shufan; Miao, Jin; Li, Shuang; Qin, Genji; Tang, Si; Li, Haoni; Gu, Hongya; Qu, Li-Jia

    2010-06-01

    1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is an important enzyme involved in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the role of the MEP pathway in plant development and metabolism, we carried out detailed analyses on a dxr mutant (GK_215C01) and two DXR transgenic co-suppression lines, OX-DXR-L2 and OX-DXR-L7. We found that the dxr mutant was albino and dwarf. It never bolted, had significantly reduced number of trichomes and most of the stomata could not close normally in the leaves. The two co-suppression lines produced more yellow inflorescences and albino sepals with no trichomes. The transcription levels of genes involved in trichome initiation were found to be strongly affected, including GLABRA1, TRANSPARENT TESTA GLABROUS 1, TRIPTYCHON and SPINDLY, expression of which is regulated by gibberellic acids (GAs). Exogenous application of GA(3) could partially rescue the dwarf phenotype and the trichome initiation of dxr, whereas exogenous application of abscisic acid (ABA) could rescue the stomata closure defect, suggesting that lower levels of both GA and ABA contribute to the phenotype in the dxr mutants. We further found that genes involved in the biosynthetic pathways of GA and ABA were coordinately regulated. These results indicate that disruption of the plastidial MEP pathway leads to biosynthetic deficiency of photosynthetic pigments, GAs and ABA, and thus the developmental abnormalities, and that the flux from the cytoplasmic mevalonate pathway is not sufficient to rescue the deficiency caused by the blockage of the plastidial MEP pathway. These results reveal a critical role for the MEP biosynthetic pathway in controlling the biosynthesis of isoprenoids.

  2. In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

    PubMed

    Bulley, Simon J; Droubi, Alaa; Clarke, Jonathan H; Anderson, Karen E; Stephens, Len R; Hawkins, Phillip T; Irvine, Robin F

    2016-09-20

    Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal. PMID:27601656

  3. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5'-Phosphate Production in E. coli.

    PubMed

    Oberhardt, Matthew A; Zarecki, Raphy; Reshef, Leah; Xia, Fangfang; Duran-Frigola, Miquel; Schreiber, Rachel; Henry, Christopher S; Ben-Tal, Nir; Dwyer, Daniel J; Gophna, Uri; Ruppin, Eytan

    2016-01-01

    Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly. PMID:26821166

  4. Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

    SciTech Connect

    Chang, Y.C.; Scott, R.D.; Graves, D.J.

    1987-01-27

    A new vitamin B/sub 6/ analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear /sup 1/H-/sup 18/F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDLP showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL except the K/sub m/ of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. /sup 19/F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the /sup 19/F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy. Results of this study indicate that the protein structure surrounding the coenzyme molecule, as well as the coenzyme configuration, is altered upon the binding of ligands.

  5. C-H activation in pyridoxal-5'-phosphate Schiff bases: the role of the imine nitrogen. A combined experimental and computational study.

    PubMed

    Casasnovas, Rodrigo; Adrover, Miquel; Ortega-Castro, Joaquin; Frau, Juan; Donoso, Josefa; Muñoz, Francisco

    2012-09-01

    The origins of C-H activation in pyridoxal-5'-phosphate (PLP) Schiff bases and modulation of reaction specificity in PLP-enzymes are still not completely understood. There are no available studies that compare the reactivity of C4' carbons in ketimine Schiff bases with that of Cα carbons in their aldimine counterparts, which is essential to unravel the mechanisms that govern the evolution of their common carbanionic intermediates. Second-order rate constants for phosphate-catalyzed proton/deuterium exchange reactions in D(2)O of C4' carbons suffer a 10(5)-fold increase due to Schiff base formation (k(B) = 5.3 × 10(1) M(-1) s(-1)) according to NMR measurements. The C4' carbon acidity is also increased to pK(a) = 9.8, which is significantly higher than that of Cα in PLP-aldimines. DFT calculations reveal the role of each heteroatom in modulating the electrophilicity of C4' and Cα carbons. Specifically, the protonation state of pyridine nitrogen is the main factor in determining the absolute carbon acidity in aldimines (pK(a) of Cα varies from ∼14 to ∼23) and ketimines (pK(a) of C4' varies from ∼12 to ∼18), whereas the protonation state of both imine nitrogen and O3' phenol oxygen modulates the relative acidities of Cα and C4' from 1.5 to 7.5 pK(a) units. Our results provide an explanation to the modulation of reaction specificity observed in different PLP-enzymes based on the differences in the protonation state of the cofactor and H-bonding patterns in the active site. PMID:22845654

  6. Genetics Home Reference: GM3 synthase deficiency

    MedlinePlus

    ... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... diagnosis or management of GM3 synthase deficiency: American Epilepsy Society: Find a Doctor Clinic for Special Children ( ...

  7. [Differences in the light-activation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and of ribulose-5-phosphate kinase between plants containing the Calvin and those containing the C4-dicarboxylic acid pathway of photosynthetic carbon reduction].

    PubMed

    Steiger, E; Ziegler, I; Ziegler, H

    1971-06-01

    1. Preceding experiments had shown that irradiance of intact leaves or of isolated chloroplasts causes a reversible increase in the activity of NADP-GPD (Ziegler and Ziegler, 1965) as well as of ribulose-5-phosphate kinase (Latzko and Gibbs, 1969). Examination of several species which carry out the Calvin type of photosynthetic CO2 fixation (Vicia faba, Spinacia oleracea, Nicotiana tabacum, Avena sativa) now revealed that the dark level of NADP-GPD activity ranges between 300-400 μmol NADPH/mg chlorophyll·h; irradiance causes an activation to an turnover rate of 900-1600 μmol NADPH/mg chlorophyll·h. 2. The dark-level of ribulose-5-phosphate kinase in these Calvin type plants corresponds to about 400 \\gmmol PO4---/mg chlorophyll\\sdh. It rises to 900\\2-1300 \\gmmol PO4---/mg chlorophyll\\sdh after irradiance. 3. In all species examined which carry out the C4-dicarboxylic acid type of CO2 fixation (Zea mays, Cyperus rotundus, Portulacca oleracea, Saccharum officinarum) the dark-level of NADP-GPD as well as of ribulose-5-phosphate kinase is already as high as the light-level of Calvin type plants. In these species irradiance either activates both enzymes only to a small extent (Saccharum officinarum, Portulacea oleracea) or it activates only one of the two enzymes to an exceptional high activity (NADP-GPD in Zea mays, ribulose-5-phosphate kinase in Cyperus rotundus), while the activity of the other one remains nearly constant. 4. The dark-level of NADP-GPD in young Zea mays (2 leaves expanded) is as high as in adult plants; moreover its further activation by light corresponds to that in adult plants. In contrast, the dark-activity of ribulose-5-phosphate kinase in young Zea mays corresponds to the lower level found in Calvin type plants and is activated by irradiance in the same manner as it is in the latter plants. 5. The activity of ribose-5-phosphate isomerase is not influenced by light.

  8. 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.

  9. The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site

    PubMed Central

    Clarke, Jonathan H.; Giudici, Maria-Luisa; Burke, John E.; Williams, Roger L.; Maloney, David J.; Marugan, Juan; Irvine, Robin F.

    2014-01-01

    NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100 μM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen–deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and β homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 μM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na+/K+-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of ‘domes’ on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or β. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks. PMID:25495341

  10. A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis.

    PubMed

    Takahashi, S; Kuzuyama, T; Watanabe, H; Seto, H

    1998-08-18

    Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli. PMID:9707569

  11. A 1-deoxy-d-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-d-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis

    PubMed Central

    Takahashi, Shunji; Kuzuyama, Tomohisa; Watanabe, Hiroyuki; Seto, Haruo

    1998-01-01

    Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-d-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-d-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-d-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-d-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-d-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli. PMID:9707569

  12. Arabidopsis 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate synthase: cDNA cloning and expression analyses.

    PubMed

    Matsuura, Keiichi; Miyagawa, Isao; Kobayashi, Masaru; Ohta, Daisaku; Matoh, Toru

    2003-07-01

    The molecular characterization of two isoforms of 3-deoxy-d-manno-oct-2-ulosonate (KDO) -8-phosphate synthase (AtkdsA1 and AtkdsA2) from Arabidopsis is reported here. First, by isolating a full-length cDNA for AtkdsA1, it was confirmed that the deduced primary structures of AtkdsA1 and AtkdsA2 proteins were 93% identical. Functional expression and purification studies demonstrated the efficient catalytic activity of the AtkdsA1 enzyme to produce KDO-8-phosphate from phosphoenolpyruvate and d-arabinose-5-phosphate. RT-PCR and RNA-gel blot analysis revealed different expression profiles for both genes; the AtkdsA1 gene was predominantly expressed in the shoots, while the AtkdsA2 transcript accumulated to a higher level in the roots, implicating differential roles of these isoforms in planta.

  13. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    PubMed Central

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2010-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to the extraordinary structural diversity of naturally occurring polyketides. PMID:19362634

  14. Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation.

    PubMed

    Walter, Michael H; Floss, Daniela S; Hans, Joachim; Fester, Thomas; Strack, Dieter

    2007-01-01

    During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C(14) mycorradicin and C(13) cyclohexenone derivatives, are predicted to originate from a common C(40) carotenoid precursor. Mycorradicin is the chromophore of the "yellow pigment" responsible for the long-known yellow discoloration of colonized roots. The biosynthesis of apocarotenoids has been investigated with a focus on the two first steps of the methylerythritol phosphate (MEP) pathway catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). In Medicago truncatula and other plants the DXS2 isogene appears to be specifically involved in the AM-mediated accumulation of apocarotenoids, whereas in the case of DXR a single gene contributes to both housekeeping and mycorrhizal (apo)carotenoid biosynthesis. Immunolocalization of DXR in mycorrhizal maize roots indicated an arbuscule-associated protein deposition, which occurs late in arbuscule development and accompanies arbuscule degeneration and breakdown. The DXS2 isogene is being developed as a tool to knock-down apocarotenoid biosynthesis in mycorrhizal roots by an RNAi strategy. Preliminary results from this approach provide starting points to suggest a new kind of function for apocarotenoids in mycorrhizal roots.

  15. Flowery odor formation revealed by differential expression of monoterpene biosynthetic genes and monoterpene accumulation in rose (Rosa rugosa Thunb.).

    PubMed

    Feng, Liguo; Chen, Chen; Li, Tinglin; Wang, Meng; Tao, Jun; Zhao, Daqiu; Sheng, Lixia

    2014-02-01

    Rosa rugosa is an important ornamental and economical plant. In this paper, four genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), alcohol acyltransferase (AAT) and linalool synthase (LIS) involved in the monoterpene biosynthesis pathways were isolated from R. rugosa 'Tangzi', and the expression patterns of these genes in different flower development stages and different parts of floral organs were determined by real-time quantitative fluorescence PCR. Furthermore, a comprehensive analysis was carried out into the relationship between expression of four monoterpene synthesis genes and accumulation of main volatile monoterpenes and their acetic acid ester derivatives. The results showed that the genes RrDXS, RrDXR and RrLIS showed consistent expressions during the development process for R. rugosa flower from budding to withering stage, the overall expression levels of gene RrDXS and RrLIS were obviously lower as compared with those of gene RrDXR and RrAAT. Although the gene RrDXS, RrDXR, RrAAT and RrLIS were expressed in all parts of R. rugosa floral organs, the expression levels varied significantly. The variations in the constituent and content of volatile monoterpenes including citronellol, geraniol, nerol, linalool, citronellyl acetate, geranyl acetate and neryl acetate at different development stages and parts of floral organs were significantly different. On this basis, we concluded that the gene RrDXR and RrAAT might play a key role in the biosynthesis of volatile monoterpenes in R. rugosa flowers, and the two genes are important candidate genes for the regulation of secondary metabolism for rose aromatic components.

  16. Flowery odor formation revealed by differential expression of monoterpene biosynthetic genes and monoterpene accumulation in rose (Rosa rugosa Thunb.).

    PubMed

    Feng, Liguo; Chen, Chen; Li, Tinglin; Wang, Meng; Tao, Jun; Zhao, Daqiu; Sheng, Lixia

    2014-02-01

    Rosa rugosa is an important ornamental and economical plant. In this paper, four genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), alcohol acyltransferase (AAT) and linalool synthase (LIS) involved in the monoterpene biosynthesis pathways were isolated from R. rugosa 'Tangzi', and the expression patterns of these genes in different flower development stages and different parts of floral organs were determined by real-time quantitative fluorescence PCR. Furthermore, a comprehensive analysis was carried out into the relationship between expression of four monoterpene synthesis genes and accumulation of main volatile monoterpenes and their acetic acid ester derivatives. The results showed that the genes RrDXS, RrDXR and RrLIS showed consistent expressions during the development process for R. rugosa flower from budding to withering stage, the overall expression levels of gene RrDXS and RrLIS were obviously lower as compared with those of gene RrDXR and RrAAT. Although the gene RrDXS, RrDXR, RrAAT and RrLIS were expressed in all parts of R. rugosa floral organs, the expression levels varied significantly. The variations in the constituent and content of volatile monoterpenes including citronellol, geraniol, nerol, linalool, citronellyl acetate, geranyl acetate and neryl acetate at different development stages and parts of floral organs were significantly different. On this basis, we concluded that the gene RrDXR and RrAAT might play a key role in the biosynthesis of volatile monoterpenes in R. rugosa flowers, and the two genes are important candidate genes for the regulation of secondary metabolism for rose aromatic components. PMID:24384414

  17. Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

    PubMed

    Gregory, Jesse F; DeRatt, Barbara N; Rios-Avila, Luisa; Ralat, Maria; Stacpoole, Peter W

    2016-07-01

    The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.

  18. 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. PMID:27576495

  19. Methylerythritol phosphate pathway to isoprenoids: kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum.

    PubMed

    Singh, Vivek Kumar; Ghosh, Indira

    2013-09-01

    The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system. This study demonstrates that both types of enzyme targets, one acting via flux reduction and the other by metabolite accumulation, exist in P. falciparum MEP pathway. These groups of targets can be exploited for independent anti-malarial drugs.

  20. Producing biofuels using polyketide synthases

    SciTech Connect

    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.

  1. Synthesis of Functionalized Cinnamaldehyde Derivatives by an Oxidative Heck Reaction and Their Use as Starting Materials for Preparation of Mycobacterium tuberculosis 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase Inhibitors

    PubMed Central

    2011-01-01

    Cinnamaldehyde derivatives were synthesized in good to excellent yields in one step by a mild and selective, base-free palladium(II)-catalyzed oxidative Heck reaction starting from acrolein and various arylboronic acids. Prepared α,β-unsaturated aldehydes were used for synthesis of novel α-aryl substituted fosmidomycin analogues, which were evaluated for their inhibition of Mycobacterium tuberculosis 1-deoxy-d-xylulose 5-phosphate reductoisomerase. IC50 values between 0.8 and 27.3 μM were measured. The best compound showed activity comparable to that of the most potent previously reported α-aryl substituted fosmidomycin-class inhibitor. PMID:21936546

  2. 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.

  3. Trichodiene synthase. Substrate specificity and inhibition.

    PubMed

    Cane, D E; Yang, G; Xue, Q; Shim, J H

    1995-02-28

    The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70. PMID:7873526

  4. 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.

  5. Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

    PubMed Central

    Richter, G; Volk, R; Krieger, C; Lahm, H W; Röthlisberger, U; Bacher, A

    1992-01-01

    3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase. Images PMID:1597419

  6. Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution.

    PubMed Central

    Clausen, T; Huber, R; Prade, L; Wahl, M C; Messerschmidt, A

    1998-01-01

    The transsulfuration enzyme cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine beta-lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP. PMID:9843488

  7. Novel structural arrangement of nematode cystathionine β-synthases: characterization of Caenorhabditis elegans CBS-1

    PubMed Central

    Vozdek, Roman; Hnízda, Aleš; Krijt, Jakub; Kostrouchová, Marta; Kožich, Viktor

    2012-01-01

    CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins. PMID:22240119

  8. COMMON BASIS FOR THE MECHANISM OF METALLO AND NON-METALLO KDO8P SYNTHASES

    PubMed Central

    Tao, Peng; Schlegel, H. Bernhard; Gatti, Domenico L.

    2010-01-01

    The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (Pi) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water. This reaction is one of the key steps in the biosynthesis of bacterial endotoxins. The evolutionary tree of KDO8PS is evenly divided between metal and non-metal forms, both having essentially identical structures. Mutagenesis and crystallographic studies suggests that one or two residues at most determine whether or not KDO8PS requires a metal for function, a clear example of “minimalist evolution”. Quantum mechanical/molecular mechanical (QM/MM) simulations of both the enzymatic and non-enzymatic synthesis of KDO8P have revealed the mechanism underlying the switch between metal and non-metal dependent catalysis. The principle emerging from these studies is that this conversion is possible in KDO8PS because the metal is not involved in an activation process, but primarily contributes to orienting properly the reactants to lower the activation energy, an action easily mimicked by amino acid side-chains. PMID:20825995

  9. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    PubMed

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  10. 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.

  11. Nitric oxide synthases in pregnant rat uterus.

    PubMed

    Farina, M; Ribeiro, M L; Franchi, A

    2001-03-01

    The conversion of [14C]arginine into [14C]citrulline as an indicator of nitric oxide synthesis was studied in uteri isolated from rats on different days of gestation, after labour and during dioestrus. Nitric oxide synthesis was present in uterine tissues isolated at each stage of gestation and also in tissues collected during dioestrus and after labour. Expression of neuronal nitric oxide synthase was not detectable at any of the stages studied. Endothelial nitric oxide synthase was present at all the stages studied, but there was a significant increase on day 13 of gestation and a decrease thereafter, with the lowest expression recorded on the day after labour. Inducible nitric oxide synthase expression in rat uteri increased substantially during pregnancy, with the highest expression on day 13 of gestation; expression decreased at term and after labour. The changes in expression of inducible nitric oxide synthase were coincident with the changes in nitric oxide synthase activity in uteri treated with aminoguanidine. Thus, these findings indicate that an increase in expression of inducible nitric oxide synthase in the uterus may be important for maintenance of uterine quiescence during pregnancy and its decrease near the time of labour could have an effect on the start of uterine contractility. PMID:11226066

  12. 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.

  13. Dihydrodipicolinate synthase from Thermotoga maritima.

    PubMed

    Pearce, F Grant; Perugini, Matthew A; McKerchar, Hannah J; Gerrard, Juliet A

    2006-12-01

    DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 degrees C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage. PMID:16872276

  14. 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

  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. Structures of Aquifex aeolicus KDO8P synthase in complex with R5P and PEP, and with a bisubstrate inhibitor: role of active site water in catalysis.

    PubMed

    Wang, J; Duewel, H S; Woodard, R W; Gatti, D L

    2001-12-25

    We have determined the crystal structures of the metalloenzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase from Aquifex aeolicus in complex with phosphoenolpyruvate (PEP) and ribose 5-phosphate (R5P), and with a bisubstrate inhibitor that mimics the postulated linear reaction intermediate. R5P, which is not a substrate for KDO8P synthase, binds in a manner similar to that of arabinose 5-phosphate (A5P), which is the natural substrate. The lack of reactivity of R5P appears to be primarily a consequence of the loss of a water molecule coordinated to Cd(2+) and located on the si side of PEP. This water molecule is no longer present because it cannot form a hydrogen bond with C2-OH(R5P), which is oriented in a different direction from C2-OH(A5P). The bisubstrate inhibitor binds with its phosphate and phosphonate moieties occupying the positions of the phosphate groups of A5P and PEP, respectively. One of the inhibitor hydroxyls replaces water as a ligand of Cd(2+). The current work supports a mechanism for the synthesis of KDO8P, in which a hydroxide ion on the si side of PEP attacks C2(PEP), forming a tetrahedral-like intermediate with a buildup of negative charge at C3(PEP). The ensuing condensation of C3(PEP) with C1(A5P) would be favored by a proton transfer from the phosphate moiety of PEP to the aldehyde carbonyl of A5P to generate the hydroxyl. Overall, the process can be described as a syn addition of water and A5P to the si side of PEP.

  17. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  18. 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

  19. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

  20. Nuclear genetic defects of mitochondrial ATP synthase.

    PubMed

    Hejzlarová, K; Mráček, T; Vrbacký, M; Kaplanová, V; Karbanová, V; Nůsková, H; Pecina, P; Houštěk, J

    2014-01-01

    Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis.

  1. An investigation into eukaryotic pseudouridine synthases.

    PubMed

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  2. Exploring biosynthetic diversity with trichodiene synthase.

    PubMed

    Vedula, L Sangeetha; Zhao, Yuxin; Coates, Robert M; Koyama, Tanetoshi; Cane, David E; Christianson, David W

    2007-10-15

    Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP(i)). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.85A resolution X-ray crystal structure of the complex with Mg(2+) (3)-PP(i) and the benzyl triethylammonium cation, which is a bulkier mimic of the bisabolyl carbocation intermediate in catalysis. Taken together, these findings show that the active site of trichodiene synthase is sufficiently flexible to accommodate bulkier and electronically-diverse substrates and intermediates, which could indicate additional potential for the biosynthetic utility of this terpenoid cyclase. PMID:17678871

  3. Cellulose Synthase Complexes: Composition and Regulation

    PubMed Central

    Lei, Lei; Li, Shundai; Gu, Ying

    2012-01-01

    Live cell imaging has greatly advanced our knowledge on the molecular mechanism by which cellulose is deposited. Both the actin and microtubule cytoskeleton are involved in assuring the proper distribution, organization, and dynamics of cellulose synthase complexes (CSCs). This review is an update on the most recent progress on the characterization of the composition, regulation, and trafficking of CSCs. With the newly identified cellulose synthase interactive protein 1 (CSI1) on hand, we begin to unveil the mystery of an intimate relationship between cellulose microfibrils and microtubules. PMID:22639663

  4. Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens.

    PubMed

    Umeda, F; Nishikawa, T; Miyasaka, H; Maeda, I; Kawase, M; Yagi, K

    2001-11-01

    Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens. PMID:11916262

  5. 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. PMID:23554321

  6. Lessons from 455 Fusarium polyketide synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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.

  8. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    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.

  9. Re-Citrate Synthase from Clostridium kluyveri Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to Si-Citrate Synthase† ▿

    PubMed Central

    Li, Fuli; Hagemeier, Christoph H.; Seedorf, Henning; Gottschalk, Gerhard; Thauer, Rudolf K.

    2007-01-01

    The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity. PMID:17400742

  10. Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1.

    PubMed

    Arimura, Gen-Ichiro; Huber, Dezene P W; Bohlmann, Jörg

    2004-02-01

    Feeding forest tent caterpillars (FTCs) induced local and systemic diurnal emissions of (-)-germacrene D, along with (E)-beta-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), benzene cyanide, and (E,E)-alpha-farnesene, from leaves of hybrid poplar. FTC feeding induced substantially higher levels of volatiles in local and systemic leaves than did mechanical wounding. A full-length poplar sesquiterpene synthase cDNA (PtdTPS1) was isolated and functionally identified as (-)-germacrene D synthase. Expression of PtdTPS1, expression of genes of early, intermediate and late steps in terpenoid biosynthesis, and expression of a lipoxygenase gene (PtdLOX1) were analyzed in local FTC-infested and systemic leaves. Transcript levels of PtdTPS1 and PtdLOX1 were strongly increased in response to herbivory. PtdTPS1 was also induced by mechanical wounding or by methyl jasmonate (MeJA) treatment. FTC feeding did not affect transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and isoprene synthase (IPS). Two other TPS genes, PtdTPS2 and PtTPS3, and farnesyl diphosphate synthase were only very transiently induced. These results illustrate differential expression of terpenoid pathway genes in response to insect feeding and a key function of (-)-germacrene D synthase PtdTPS1 for herbivore-induced local and systemic volatile emissions in hybrid poplar. FTC-induced transcripts of PtdTPS1 followed diurnal rhythm. Spatial patterns of FTC-induced PtdTPS1 transcript accumulation revealed acropetal but not basipetal direction of the systemic response. Implications for tritrophic poplar-FTC-predator/parasitoid interactions are discussed. PMID:14756770

  11. Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

    PubMed

    Strambini, G B; Cioni, P; Peracchi, A; Mozzarelli, A

    1992-08-25

    Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

    PubMed Central

    Darimont, B.; Stehlin, C.; Szadkowski, H.; Kirschner, K.

    1998-01-01

    Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184. PMID:9605328

  13. 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.

  14. The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT): effects of the small subunits and insights from bacterial mimics of human hLCB2a HSAN1 mutations.

    PubMed

    Beattie, Ashley E; Gupta, Sita D; Frankova, Lenka; Kazlauskaite, Agne; Harmon, Jeffrey M; Dunn, Teresa M; Campopiano, Dominic J

    2013-01-01

    The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form. PMID:24175284

  15. The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT): effects of the small subunits and insights from bacterial mimics of human hLCB2a HSAN1 mutations.

    PubMed

    Beattie, Ashley E; Gupta, Sita D; Frankova, Lenka; Kazlauskaite, Agne; Harmon, Jeffrey M; Dunn, Teresa M; Campopiano, Dominic J

    2013-01-01

    The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form.

  16. 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.

  17. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    PubMed

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  18. Building-block selectivity of polyketide synthases.

    PubMed

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  19. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    PubMed

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

    2014-12-26

    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.

  20. 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

  1. All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity[S

    PubMed Central

    Ding, Tingbo; Kabir, Inamul; Li, Yue; Lou, Caixia; Yazdanyar, Amirfarbod; Xu, Jiachen; Dong, Jibin; Zhou, Hongwen; Park, Taesik; Boutjdir, Mohamed; Li, Zhiqiang; Jiang, Xian-Cheng

    2015-01-01

    Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo. PMID:25605874

  2. 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.

  3. 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.

  4. 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.

  5. Regulation of mitochondrial ATP synthase in cardiac pathophysiology.

    PubMed

    Long, Qinqiang; Yang, Kevin; Yang, Qinglin

    2015-01-01

    Mitochondrial function is paramount to energy homeostasis, metabolism, signaling, and apoptosis in cells. Mitochondrial complex V (ATP synthase), a molecular motor, is the ultimate ATP generator and a key determinant of mitochondrial function. ATP synthase catalyzes the final coupling step of oxidative phosphorylation to supply energy in the form of ATP. Alterations at this step will crucially impact mitochondrial respiration and hence cardiac performance. It is well established that cardiac contractility is strongly dependent on the mitochondria, and that myocardial ATP depletion is a key feature of heart failure. ATP synthase dysfunction can cause and exacerbate human diseases, such as cardiomyopathy and heart failure. While ATP synthase has been extensively studied, essential questions related to how the regulation of ATP synthase determines energy metabolism in the heart linger and therapies targeting this important mechanism remain scarce. This review will visit the main findings, identify unsolved issues and provide insights into potential future perspectives related to the regulation of ATP synthase and cardiac pathophysiology.

  6. Surrogate splicing for functional analysis of sesquiterpene synthase genes.

    PubMed

    Wu, Shuiqin; Schoenbeck, Mark A; Greenhagen, Bryan T; Takahashi, Shunji; Lee, Sungbeom; Coates, Robert M; Chappell, Joseph

    2005-07-01

    A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing beta-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for alpha-barbatene, thujopsene, and beta-chamigrene biosynthesis. PMID:15965019

  7. 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.

  8. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    PubMed

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds. PMID:15464152

  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.; Itoh, Aya

    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. Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase.

    PubMed

    Oven, Matjaz; Page, Jonathan E; Zenk, Meinhart H; Kutchan, Toni M

    2002-02-15

    The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451-31459). PMID:11706029

  12. Engineering of chimeric class II polyhydroxyalkanoate synthases.

    PubMed

    Niamsiri, Nuttawee; Delamarre, Soazig C; Kim, Young-Rok; Batt, Carl A

    2004-11-01

    PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic

  13. 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. PMID:26936952

  14. 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.

  15. 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

  16. Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors.

    PubMed

    Botella-Pavía, Patricia; Besumbes, Oscar; Phillips, Michael A; Carretero-Paulet, Lorenzo; Boronat, Albert; Rodríguez-Concepción, Manuel

    2004-10-01

    Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.

  17. 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.

  18. Activities and regulation of peptidoglycan synthases

    PubMed Central

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

    2015-01-01

    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. PMID:26370943

  19. ATP synthase: a tentative structural model.

    PubMed

    Engelbrecht, S; Junge, W

    1997-09-15

    Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer. PMID:9323021

  20. Torque generation mechanism of ATP synthase

    NASA Astrophysics Data System (ADS)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  1. ATP synthases from archaea: the beauty of a molecular motor.

    PubMed

    Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed.

  2. [Four cases of aldosterone synthase deficiency in childhood].

    PubMed

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  3. 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

  4. Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases.

    PubMed

    Aaron, Julie A; Christianson, David W

    2010-01-01

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. 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

  7. Pseudouridines and pseudouridine synthases of the ribosome.

    PubMed

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  8. Hyaluronan synthases and hyaluronidases in nasal polyps.

    PubMed

    Panogeorgou, T; Tserbini, E; Filou, S; Vynios, D H; Naxakis, S S; Papadas, T A; Goumas, P D; Mastronikolis, N S

    2016-07-01

    Nasal polyps (NPs) are benign lesions of nasal and paranasal sinuses mucosa affecting 1-4 % of all adults. Nasal polyposis affects the quality of patient's life as it causes nasal obstruction, postnasal drainage, purulent nasal discharge, hyposmia or anosmia, chronic sinusitis, facial pain and snoring. Without treatment, the disease can alter the craniofacial skeleton in cases of extended growth of polyps. The development of NPs is caused by the hyperplasia of nasal or paranasal sinuses mucosa, and edema of extracellular matrix. This is usually the result of high concentration of high molecular mass hyaluronan (HA) which is either overproduced or accumulated from blood supply. The size of HA presents high diversity and, especially in pathologic conditions, chains of low molecular mass can be observed. In NPs, chains of about 200 kDa have been identified and considered to be responsible for the inflammation. The purpose of the present study was the investigation, in NPs and normal nasal mucosa (NM), of the expression of the wild-type and alternatively spliced forms of hyaluronidases, their immunolocalization, and the expression of HA synthases to examine the isoform(s) responsible for the increased amounts of HA in NPs. Hyaluronidases' presence was examined on mRNA (RT-PCR analysis) and protein (immunohistochemistry) levels. Hyaluronan synthases' presence was examined on mRNA levels. Hyaluronidases were localized in the cytoplasm of epithelial and inflammatory cells, as well as in the matrix. On mRNA level, it was found that hyal-1-wt was decreased in NPs compared to NM and hyal-1-v3, -v4 and -v5 were substantially increased. Moreover, HAS2 and HAS3 were the only hyaluronan synthases detected, the expression of which was almost similar in NPs and NM. Overall, the results of the present study support that hyaluronidases are the main enzymes responsible for the decreased size of hyaluronan observed in NPs; thus they behave as inflammatory agents. Therefore, they

  9. Structure of Aminodeoxychorismate Synthase from Stenotrophomonas maltophilia†

    PubMed Central

    Bera, Asim K.; Atanasova, Vesna; Dhanda, Anjali; Ladner, Jane E.; Parsons, James F.

    2012-01-01

    PabB, aminodeoxychorismate synthase, is the chorismic acid binding component of the heterodimeric PabAB complex that converts chorismic acid to 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folic acid in microorganisms. The second component, a glutamine amidotransferase subunit, PabA, generates ammonia that is channeled to the PabB active site where it attacks the C4 carbon of a chorismate derived intermediate that is covalently bound, through C2, to an active site lysine residue. The presence of a PIKGT motif was, until recently, believed to be discriminate PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active site motif and does not form a covalent enzyme-substrate intermediate with chorismate. A subclass of PabB enzymes that employ an alternative mechanism requiring two equivalents of ammonia from glutamine and that feature a noncovalently bound 2-amino-2-deoxyisochorismate intermediate was recently identified. Here we report the 2.25 Å crystal structure of PabB from the emerging pathogen Stenotrophomonas maltophilia. It is the first reported structure of a PabB that features the PIAGT motif. Surprisingly, no dedicated pabA is evident in the genome of S. maltophilia suggesting that another cellular amidotransferase is able to fulfill the role of PabA in this organism. Evaluation of the ammonia-dependent aminodeoxychorismate synthase activity of S. maltophilia PabB alone revealed that it is virtually inactive. However, in the presence of a heterologous PabA surrogate, typical levels of activity were observed using either glutamine or ammonia as the nitrogen source. Additionally, the structure suggests that a key segment of the polypeptide can remodel itself to interact with a nonspecialized or shared amidotransferase partner in vivo. The structure and mass spectral analysis further suggest that S. maltophilia PabB, like Escherichia coli PabB, binds tryptophan in a vestigial regulatory site

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

    PubMed

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

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. PMID:26392384

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

    PubMed

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

    2015-11-20

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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    PubMed

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  17. Prenyltransferases of the dimethylallyltryptophan synthase superfamily.

    PubMed

    Yu, Xia; Li, Shu-Ming

    2012-01-01

    Prenylated natural products often have interesting biological and pharmacological activities clearly distinct from their nonprenylated precursors. Prenyltransferases are responsible for the attachment of prenyl moieties to a number of acceptors and contribute significantly to structural and biological diversity of these compounds in nature. In the past 8 years, significant progress has been achieved in the molecular biological, biochemical, and structural biological investigation of the prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily. These soluble enzymes are involved in the biosynthesis of fungal secondary metabolites and mainly catalyze prenylation of diverse indole derivatives, including tryptophan and tryptophan-containing cyclic dipeptides. The members of the DMATS superfamily show promising flexibility toward their aromatic substrates and catalyze highly regio- and stereoselective prenyltransfer reactions. These features were successfully used for chemoenzymatic synthesis, not only for production of prenylated simple indoles and cyclic dipeptides but also for prenylated hydroxynaphthalenes and flavonoids, which are usually found in bacteria and plants, respectively.

  18. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    PubMed

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  19. 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

  20. Role of cysteine residues in pseudouridine synthases of different families.

    PubMed

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  1. Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

    PubMed Central

    Breda, Ardala; Martinelli, Leonardo K. B.; Bizarro, Cristiano V.; Rosado, Leonardo A.; Borges, Caroline B.; Santos, Diógenes S.; Basso, Luiz A.

    2012-01-01

    The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of Mt

  2. Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

    PubMed

    Breda, Ardala; Martinelli, Leonardo K B; Bizarro, Cristiano V; Rosado, Leonardo A; Borges, Caroline B; Santos, Diógenes S; Basso, Luiz A

    2012-01-01

    The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of

  3. Structural basis for competitive inhibition of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Vibrio cholerae.

    PubMed

    Islam, Zeyaul; Kumar, Adarsh; Singh, Suruchi; Salmon, Laurent; Karthikeyan, Subramanian

    2015-05-01

    The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate. Moreover, DHBPS is shown to be indispensable for Mycobacterium, Salmonella, and Helicobacter species. Despite the essentiality of this enzyme in bacteria, no inhibitor has been identified hitherto. Here, we describe kinetic and crystal structure characterization of DHBPS from Vibrio cholerae (vDHBPS) with a competitive inhibitor 4-phospho-d-erythronohydroxamic acid (4PEH) at 1.86-Å resolution. In addition, we also report the structural characterization of vDHBPS in its apo form and in complex with its substrate and substrate plus metal ions at 1.96-, 1.59-, and 2.04-Å resolution, respectively. Comparison of these crystal structures suggests that 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity. Furthermore, vDHBPS structures complexed with substrate and metal ions reveal that, unlike Candida albicans, binding of substrate to vDHBPS induces a conformational change from an open to closed conformation. Interestingly, the position of second metal ion, which is different from that of Methanococcus jannaschii, strongly supports an active role in the catalytic mechanism. Thus, the kinetic and structural characterization of vDHBPS reveals the molecular mechanism of inhibition shown by 4PEH and that it can be explored further for designing novel antibiotics.

  4. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    SciTech Connect

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  5. Cloning Rosa hybrid phenylacetaldehyde synthase for the production of 2-phenylethanol in a whole cell Escherichia coli system.

    PubMed

    Achmon, Yigal; Ben-Barak Zelas, Zohar; Fishman, Ayelet

    2014-04-01

    2-Phenylethanol (2-PE) is a desirable compound in the food and perfumery industries with a characteristic rose fragrance. Until now, most of the studied biotechnological processes to produce 2-PE were conducted using natural 2-PE-producing yeasts. Only several researches were conducted in other genetically engineered microorganisms that simulated the Ehrlich pathway for the conversion of amino acids to fusel alcohols. Here, a novel metabolic pathway has been designed in Escherichia coli to produce 2-PE, using the Rosa hybrid phenylacetaldehyde synthase (PAAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme capable of transforming L-phenylalanine (L-phe) into phenylacetaldehyde by decarboxylation and oxidation. To overcome the enzyme insolubility in E. coli, several plasmids and host strains were tested for their expression ability. The desired results were obtained by using the pTYB21 plasmid containing the intein tag from the Saccharomyces cerevisiae VMA1. It was discovered that the intein PAAS activity is temperature-dependent, working well in the range of 25 to 30 °C but losing most of its activity at 37 °C. When external PLP cofactor was added, the cells produced 0.39 g l⁻¹ 2-PE directly from L-phe. In addition, a biotransformation that was based only on internal de novo PLP synthesis produced 0.34 g l⁻¹ 2-PE, thus creating for the first time an E. coli strain that can produce 2-PE from L-phe without the need for exterior cofactor additions.

  6. Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction.

    PubMed

    Stojanovski, Bosko M; Ferreira, Gloria C

    2015-12-25

    5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.

  7. The redox behavior of the heme in cystathionine beta-synthase is sensitive to pH.

    PubMed

    Pazicni, Samuel; Lukat-Rodgers, Gudrun S; Oliveriusová, Jana; Rees, Katherine A; Parks, Ryan B; Clark, Robert W; Rodgers, Kenton R; Kraus, Jan P; Burstyn, Judith N

    2004-11-23

    Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated. PMID:15544339

  8. The Catalytic and Conformational Cycle of Aquifex aeolicus KDO8P Synthase: Role of the L7 Loop†,‡

    PubMed Central

    Xu, Xingjue; Kona, Fathima; Wang, Jian; Lu, Jinshuang; Stemmler, Timothy; Gatti, Domenico L.

    2015-01-01

    KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (Pi). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg106 side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller KmPEP, larger KiA5P and KmA5P) and the product side (smaller KiPi and KiKDO8P) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by Pi and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme. PMID:16156656

  9. The Pseudouridine Synthases Proceed through a Glycal Intermediate

    PubMed Central

    2016-01-01

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2′. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases. PMID:27292228

  10. Colorimetric Coupled Enzyme Assay for Cystathionine β-Synthase.

    PubMed

    Rocchiccioli, Marco; Moschini, Roberta; Cappiello, Laura; Balestri, Francesco; Cappiello, Mario; Mura, Umberto; Del-Corso, Antonella

    2016-01-01

    A colorimetric coupled enzyme assay for the determination of cystathionine β-synthase activity is described. The method exploits cystathionine γ-lyase as an ancillary enzyme capable of transforming cystathionine, produced by cystathionine β-synthase, into cysteine. The cysteine is then spectrophotometrically detected at 560 nm, after its specific complexation with ninhydrin. This method was used to detect cystathionine β-synthase in crude extracts, and for the kinetic characterization of the enzyme partially purified from bovine kidney. A rapid two-step protocol is described for the partial purification of cystathionine γ-lyase from bovine kidney, aimed at a suitable and stable ancillary enzyme preparation. PMID:27506718

  11. Computational design and selections for an engineered, thermostable terpene synthase

    PubMed Central

    Diaz, Juan E; Lin, Chun-Shi; Kunishiro, Kazuyoshi; Feld, Birte K; Avrantinis, Sara K; Bronson, Jonathan; Greaves, John; Saven, Jeffery G; Weiss, Gregory A

    2011-01-01

    Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact proteins. Unlike the wild-type enzyme, the mutant TEAS retains enzymatic activity at 65°C. The thermostable terpene synthase variant denatures above 80°C, approximately twice the temperature of the wild-type enzyme. PMID:21739507

  12. The Pseudouridine Synthases Proceed through a Glycal Intermediate.

    PubMed

    Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G

    2016-06-29

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.

  13. 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. PMID:27480686

  14. 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.

  15. Elicitor induced activation of the methylerythritol phosphate pathway toward phytoalexins biosynthesis in rice.

    PubMed

    Okada, Atsushi; Shimizu, Takafumi; Okada, Kazunori; Kuzuyama, Tomohisa; Koga, Jinichiro; Shibuya, Naoto; Nojiri, Hideaki; Yamane, Hisakazu

    2007-09-01

    Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants.

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

    PubMed Central

    Meisler, M H; Carter, M L

    1980-01-01

    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. Images PMID:6930671

  17. 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

  18. Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

    PubMed

    Tan, A W; Nuttall, F Q

    1976-08-12

    Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.

  19. 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

  20. Linking pseudouridine synthases to growth, development and cell competition.

    PubMed

    Tortoriello, Giuseppe; de Celis, José F; Furia, Maria

    2010-08-01

    Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

  1. Citrate synthase from the liver fluke Fasciola hepatica.

    PubMed

    Zinsser, Veronika L; Moore, Catherine M; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2013-06-01

    Citrate synthase catalyses the first step of the Krebs' tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

  2. Membrane localization and topology of leukotriene C4 synthase.

    PubMed

    Christmas, Peter; Weber, Brittany M; McKee, Mary; Brown, Dennis; Soberman, Roy J

    2002-08-01

    Leukotriene C(4) (LTC(4)) synthase conjugates LTA(4) with GSH to form LTC(4). Determining the site of LTC(4) synthesis and the topology of LTC(4) synthase may uncover unappreciated intracellular roles for LTC(4), as well as how LTC(4) is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC(4) synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC(4) synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC(4) synthase. The active site of LTC(4) synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB(4) and LTC(4) occurs in different subcellular locations and suggests that LTC(4) must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane.

  3. 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

  4. 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. PMID:24040370

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    PubMed

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

  10. Reconstitution of Diphthine Synthase Activity In Vitro

    PubMed Central

    Zhu, Xuling; Kim, Jungwoo; Su, Xiaoyang; Lin, Hening

    2010-01-01

    Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). Although diphthamide modification was discovered three decades ago, in vitro reconstitution of diphthamide biosynthesis using purified proteins has not been reported. The proposed biosynthesis pathway of diphthamide involves three steps. Our laboratory has recently showed that in Pyrococcus horikoshii (P. horikoshii), the first step uses an [4Fe-4S] enzyme PhDph2 to generate a 3-amino-3-carboxypropyl radical from S-adenosyl-L-methionine (SAM) to form a C-C bond. The second step is the trimethylation of an amino group to form the diphthine intermediate. This step is catalyzed by a methyltransferase called diphthine synthase or Dph5. Here we report the in vitro reconstitution of the second step using P. horikoshii Dph5 (PhDph5). Our results demonstrate that PhDph5 is sufficient to catalyze the mono-, di-, and trimethylation of P. horikoshii EF2 (PhEF2). Interestingly, the trimethylated product from PhDph5-catalyzed reaction can easily eliminate the trimethylamino group. The potential implication of this unexpected finding on the diphthamide biosynthesis pathway is discussed. PMID:20873788

  11. Protein preparation, crystallization and preliminary X-ray analysis of Polygonum cuspidatum bifunctional chalcone synthase/benzalacetone synthase.

    PubMed

    Lu, Heshu; Yang, Mingfeng; Liu, Chunmei; Lu, Ping; Cang, Huaixing; Ma, Lanqing

    2013-08-01

    The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution. PMID:23908031

  12. 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.

  13. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    PubMed

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  14. Structural and functional organization of the animal fatty acid synthase.

    PubMed

    Smith, Stuart; Witkowski, Andrzej; Joshi, Anil K

    2003-07-01

    The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase. Each subunit contains three N-terminal domains, the beta-ketoacyl synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thiosterase. The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by beta-ketoacyl synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product. Only in the fatty acid synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for fatty acid synthesis at the two acyl carrier protein centers. Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric fatty acid synthases carrying different mutations on each subunit. Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein. The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and beta-ketoacyl synthase domains of either subunit. In contrast, the reactions involved in processing of the beta-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit. These findings suggest a revised model for the fatty acid synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between

  15. 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

  16. 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

  17. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

    PubMed

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  18. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

    PubMed Central

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A.; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  19. Mathematical modelling of the diurnal regulation of the MEP pathway in Arabidopsis.

    PubMed

    Pokhilko, Alexandra; Bou-Torrent, Jordi; Pulido, Pablo; Rodríguez-Concepción, Manuel; Ebenhöh, Oliver

    2015-05-01

    Isoprenoid molecules are essential elements of plant metabolism. Many important plant isoprenoids, such as chlorophylls, carotenoids, tocopherols, prenylated quinones and hormones are synthesised in chloroplasts via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Here we develop a mathematical model of diurnal regulation of the MEP pathway in Arabidopsis thaliana. We used both experimental and theoretical approaches to integrate mechanisms potentially involved in the diurnal control of the pathway. Our data show that flux through the MEP pathway is accelerated in light due to the photosynthesis-dependent supply of metabolic substrates of the pathway and the transcriptional regulation of key biosynthetic genes by the circadian clock. We also demonstrate that feedback regulation of both the activity and the abundance of the first enzyme of the MEP pathway (1-deoxy-D-xylulose 5-phosphate synthase, DXS) by pathway products stabilizes the flux against changes in substrate supply and adjusts the flux according to product demand under normal growth conditions. These data illustrate the central relevance of photosynthesis, the circadian clock and feedback control of DXS for the diurnal regulation of the MEP pathway.

  20. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

    PubMed

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.

  1. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    PubMed Central

    Balabaskaran Nina, Praveen; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F1 sector catalyzes ATP synthesis, whereas the Fo sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F1 and Fo sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the Fo sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a

  2. 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

  3. Plasticity and Evolution of (+)-3-Carene Synthase and (−)-Sabinene Synthase Functions of a Sitka Spruce Monoterpene Synthase Gene Family Associated with Weevil Resistance*

    PubMed Central

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

    2014-01-01

    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. PMID:25016016

  4. 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

  5. Dimers of mitochondrial ATP synthase form the permeability transition pore

    PubMed Central

    Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D.; Petronilli, Valeria; Zoratti, Mario; Szabó, Ildikó; Lippe, Giovanna; Bernardi, Paolo

    2013-01-01

    Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

  6. The ATP synthase: the understood, the uncertain and the unknown.

    PubMed

    Walker, John E

    2013-02-01

    The ATP synthases are multiprotein complexes found in the energy-transducing membranes of bacteria, chloroplasts and mitochondria. They employ a transmembrane protonmotive force, Δp, as a source of energy to drive a mechanical rotary mechanism that leads to the chemical synthesis of ATP from ADP and Pi. Their overall architecture, organization and mechanistic principles are mostly well established, but other features are less well understood. For example, ATP synthases from bacteria, mitochondria and chloroplasts differ in the mechanisms of regulation of their activity, and the molecular bases of these different mechanisms and their physiological roles are only just beginning to emerge. Another crucial feature lacking a molecular description is how rotation driven by Δp is generated, and how rotation transmits energy into the catalytic sites of the enzyme to produce the stepping action during rotation. One surprising and incompletely explained deduction based on the symmetries of c-rings in the rotor of the enzyme is that the amount of energy required by the ATP synthase to make an ATP molecule does not have a universal value. ATP synthases from multicellular organisms require the least energy, whereas the energy required to make an ATP molecule in unicellular organisms and chloroplasts is higher, and a range of values has been calculated. Finally, evidence is growing for other roles of ATP synthases in the inner membranes of mitochondria. Here the enzymes form supermolecular complexes, possibly with specific lipids, and these complexes probably contribute to, or even determine, the formation of the cristae.

  7. 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. PMID:19400802

  8. Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences

    PubMed Central

    Carroll, Andrew; Specht, Chelsea D.

    2011-01-01

    The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments. PMID:22629257

  9. SbnG, a Citrate Synthase in Staphylococcus aureus

    PubMed Central

    Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; Heinrichs, David E.; Murphy, Michael E. P.

    2014-01-01

    In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. PMID:25336653

  10. Evolutionary history of the chitin synthases of eukaryotes.

    PubMed

    Morozov, Alexey A; Likhoshway, Yelena V

    2016-06-01

    Chitin synthases are widespread among eukaryotes and known to have a complex evolutionary history in some of the groups. We have reconstructed the chitin synthase phylogeny using the most taxonomically comprehensive dataset currently available and have shown the presence of independently formed paralogous groups in oomycetes, ciliates, fungi, and all diatoms except raphid pennates. There were also two cases of horizontal gene transfer (HGT): transfer from fungus to early diatoms gave rise to diatom paralogous group, while transfer from raphid pennate diatom to Acantamoeba ancestor is, to our knowledge, restricted to a single gene in amoeba. Early evolution of chitin synthases is heavily obscured by paralogy, and further sequencing effort is necessary. PMID:26887391

  11. A functional map of the nopaline synthase promoter.

    PubMed Central

    Shaw, C H; Carter, G H; Watson, M D; Shaw, C H

    1984-01-01

    This paper describes the first functional map of a promoter expressed from the plant chromosome. We have constructed a series of overlapping deletion mutants within the region upstream of the Ti-plasmid encoded nopaline synthase (nos) gene. By monitoring nos expression in tumour tissue we have inferred a functional map of the nos promoter. The maximum length of sequence upstream of the transcription initiation point required to express wild type levels of nopaline synthase is 88 bp. Within this region, the "CAAT" box is essential for maximal activity; deletion of this sequence reduced apparent nos expression by over 80%. Presence of an intact or partial "TATA" box in the absence of the "CAAT" box supports a barely detectable level of nopaline synthase. Removal of all sequences upstream of the nos coding sequence results in no detectable activity. PMID:6493982

  12. 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.

  13. 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.

  14. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases.

    PubMed

    Lim, Yan Ping; Go, Maybelle K; Yew, Wen Shan

    2016-01-01

    Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development. PMID:27338328

  15. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    PubMed

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-26

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

  16. 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

  17. Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952.

    PubMed

    Ghimire, Gopal Prasad; Oh, Tae-Jin; Liou, Kwangkyoung; Sohng, Jae Kyung

    2008-10-31

    We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA). PMID:18612244

  18. 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. PMID:27446151

  19. PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots.

    PubMed

    Yang, Dongfeng; Ma, Pengda; Liang, Xiao; Wei, Zheng; Liang, Zongsuo; Liu, Yan; Liu, Fenghua

    2012-10-01

    Tanshinones, a group of active ingredients in Salvia miltiorrhiza, are derived from at least two biosynthetic pathways, which are the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastids. Abscisic acid (ABA) and methyl jasmonate (MJ) are two well-known plant hormones induced by water stress. In this study, effects of polyethylene glycol (PEG), ABA and MJ on tanshinone production in S. miltiorrhiza hairy roots were investigated, and the role of MJ in PEG- and ABA-induced tanshinone production was further elucidated. The results showed that tanshinone production was significantly enhanced by treatments with PEG, ABA and MJ. The mRNA levels of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS), as well as the enzyme activities of HMGR and DXS were stimulated by all three treatments. PEG and ABA triggered MJ accumulation. Effects of PEG and ABA on tanshinone production were completely abolished by the ABA biosynthesis inhibitor [tungstate (TUN)] and the MJ biosynthesis inhibitor [ibuprofen (IBU)], while effects of MJ were almost unaffected by TUN. In addition, MJ-induced tanshinone production was completely abolished by the MEP pathway inhibitor [fosmidomycin (FOS)], but was just partially arrested by the MVA pathway inhibitor [mevinolin (MEV)]. In conclusion, a signal transduction model was proposed that exogenous applications of PEG and ABA triggered endogenous MJ accumulation by activating ABA signaling pathway to stimulate tanshinone production, while exogenous MJ could directly induce tanshinone production mainly via the MEP pathway in S. miltiorrhiza hairy roots.

  20. 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- ...

  1. Incremental truncation of PHA synthases results in altered product specificity.

    PubMed

    Wang, Qian; Xia, Yongzhen; Chen, Quan; Qi, Qingsheng

    2012-05-10

    PHA synthase is the key enzyme involved in the biosynthesis of microbial polymers, polyhydroxyalkanoates (PHA). In this study, we created a hybrid library of PHA synthase gene with different crossover points by an incremental truncation method between the C-terminal fragments of the phaC(Cn) (phaC from Cupriavidus necator) and the N-terminal fragments of the phaC1(Pa) (phaC from Pseudomonas aeruginosa). As the truncation of the hybrid enzyme increased, the in vivo PHB synthesis ability of the hybrids declined gradually. PHA synthase PhaC(Cn) with a deletion on N-terminal up to 83 amino acid residues showed no synthase activity. While with the removal of up to 270 amino acids from the N-terminus, the activity of the truncated PhaC(Cn) could be complemented by the N-terminus of PhaC1(Pa). Three of the hybrid enzymes W188, W235 and W272 (named by the deleted nucleic acid number) were found to have altered product specificities. PMID:22500895

  2. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    PubMed

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  3. 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

  4. A particular phenotype in a girl with aldosterone synthase deficiency.

    PubMed

    Williams, Tracy A; Mulatero, Paolo; Bosio, Maurizio; Lewicka, Sabina; Palermo, Mario; Veglio, Franco; Armanini, Decio

    2004-07-01

    Aldosterone synthase deficiency (ASD) usually presents in infancy as a life-threatening electrolyte imbalance. A 4-wk-old child of unrelated parents was examined for failure to thrive and salt-wasting. Notable laboratory findings were hyperkalemia, high plasma renin, and low-normal aldosterone levels. Urinary metabolite ratios of corticosterone/18-hydroxycorticosterone and 18-hydroxycorticosterone/aldosterone were intermediate between ASD type I and type II. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (P450c11AS), revealed that the patient was a compound heterozygote carrying a previously described mutation located in exon 4 causing a premature stop codon (E255X) and a further, novel mutation in exon 5 that also causes a premature stop codon (Q272X). The patient's unaffected father was a heterozygous carrier of the E255X mutation, whereas the unaffected mother was a heterozygous carrier of the Q272X mutation. Therefore, the patient's CYP11B2 encodes two truncated forms of aldosterone synthase predicted to be inactive because they lack critical active site residues as well as the heme-binding site. This case of ASD is of particular interest because despite the apparent lack of aldosterone synthase activity, the patient displays low-normal aldosterone levels, thus raising the question of its source. PMID:15240589

  5. Lipoxin synthase activity of human platelet 12-lipoxygenase.

    PubMed Central

    Romano, M; Chen, X S; Takahashi, Y; Yamamoto, S; Funk, C D; Serhan, C N

    1993-01-01

    Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity. Images Figure 1 PMID:8250832

  6. Polyhydroyxalkanoate synthase fusions as a strategy for oriented enzyme immobilisation.

    PubMed

    Hooks, David O; Venning-Slater, Mark; Du, Jinping; Rehm, Bernd H A

    2014-01-01

    Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications. PMID:24962396

  7. 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.

  8. Mechanism of the beta-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase.

    PubMed

    Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2002-09-01

    The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form

  9. Detailed characterization of the substrate specificity of mouse wax synthase.

    PubMed

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  10. 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.

  11. 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.

  12. 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. PMID:24078339

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  14. Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

    PubMed

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases. PMID:25230152

  15. Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.

    PubMed

    Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A; Uversky, Vladimir N; Ferreira, Gloria C

    2014-12-01

    5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.

  16. Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.

    PubMed

    Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A; Uversky, Vladimir N; Ferreira, Gloria C

    2014-12-01

    5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme. PMID:25240868

  17. UV-B modulates the interplay between terpenoids and flavonoids in peppermint (Mentha x piperita L.).

    PubMed

    Dolzhenko, Yuliya; Bertea, Cinzia M; Occhipinti, Andrea; Bossi, Simone; Maffei, Massimo E

    2010-08-01

    Modulation of secondary metabolites by UV-B involves changes in gene expression, enzyme activity and accumulation of defence metabolites. After exposing peppermint (Mentha x piperita L.) plants grown in field (FP) and in a growth chamber (GCP) to UV-B irradiation, we analysed by qRT-PCR the expression of genes involved in terpenoid biosynthesis and encoding: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (Mds), isopentenyl diphosphate isomerase (Ippi), geranyl diphosphate synthase (Gpps), (-)-limonene synthase (Ls), (-)-limonene-3-hydroxylase (L3oh), (+)-pulegone reductase (Pr), (-)-menthone reductase (Mr), (+)-menthofuran synthase (Mfs), farnesyl diphosphate synthase (Fpps) and a putative sesquiterpene synthase (S-TPS). GCP always showed a higher terpenoid content with respect to FP. We found that in both FP and GCP, most of these genes were regulated by the UV-B treatment. The amount of most of the essential oil components, which were analysed by gas chromatography-mass spectrometry (GC-MS), was not correlated to gene expression. The total phenol composition was found to be always increased after UV-B irradiation; however, FP always showed a higher phenol content with respect to GCP. Liquid chromatography-mass spectrometry (LC-ESI-MS/MS) analyses revealed the presence of UV-B absorbing flavonoids such as eriocitrin, hesperidin, and kaempferol 7-O-rutinoside whose content significantly increased in UV-B irradiated FP, when compared to GCP. The results of this work show that UV-B irradiation differentially modulates the expression of genes involved in peppermint essential oil biogenesis and the content of UV-B absorbing flavonoids. Plants grown in field were better adapted to increasing UV-B irradiation than plants cultivated in growth chambers. The interplay between terpenoid and phenylpropanoid metabolism is also discussed.

  18. 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

  19. 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

  20. Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.

    PubMed

    Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji; Tsuge, Takeharu

    2014-05-01

    In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc.

  1. S-sulfocysteine synthase function in sensing chloroplast redox status

    PubMed Central

    Gotor, Cecilia; Romero, Luis C.

    2013-01-01

    The minor chloroplastic O-acetylserine(thiol)lyase isoform encoded by the CS26 gene in Arabidopsis thaliana has been described as an S-sulfocysteine synthase enzyme that plays an important role in chloroplast function. This enzyme is located in the thylakoid lumen, and its S-sulfocysteine activity is essential for the proper photosynthetic performance of the chloroplast under long-day growth conditions. Based on the present knowledge of this enzyme, we suggest that S-sulfocysteine synthase functions as a protein sensor to detect the accumulation of thiosulfate as a result of the inadequate detoxification of reactive oxygen species generated under conditions of excess light to produce the S-sulfocysteine molecule that triggers protection mechanisms of the photosynthetic apparatus. PMID:23333972

  2. 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

  3. Visualization of cellulose synthases in Arabidopsis secondary cell walls.

    PubMed

    Watanabe, Y; Meents, M J; McDonnell, L M; Barkwill, S; Sampathkumar, A; Cartwright, H N; Demura, T; Ehrhardt, D W; Samuels, A L; Mansfield, S D

    2015-10-01

    Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls. PMID:26450210

  4. 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

  5. S-sulfocysteine synthase function in sensing chloroplast redox status.

    PubMed

    Gotor, Cecilia; Romero, Luis C

    2013-03-01

    The minor chloroplastic O-acetylserine(thiol)lyase isoform encoded by the CS26 gene in Arabidopsis thaliana has been described as an S-sulfocysteine synthase enzyme that plays an important role in chloroplast function. This enzyme is located in the thylakoid lumen, and its S-sulfocysteine activity is essential for the proper photosynthetic performance of the chloroplast under long-day growth conditions. Based on the present knowledge of this enzyme, we suggest that S-sulfocysteine synthase functions as a protein sensor to detect the accumulation of thiosulfate as a result of the inadequate detoxification of reactive oxygen species generated under conditions of excess light to produce the S-sulfocysteine molecule that triggers protection mechanisms of the photosynthetic apparatus.

  6. Visualization of cellulose synthases in Arabidopsis secondary cell walls.

    PubMed

    Watanabe, Y; Meents, M J; McDonnell, L M; Barkwill, S; Sampathkumar, A; Cartwright, H N; Demura, T; Ehrhardt, D W; Samuels, A L; Mansfield, S D

    2015-10-01

    Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls.

  7. Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the 'yellow pigment' and other apocarotenoids.

    PubMed

    Walter, M H; Fester, T; Strack, D

    2000-03-01

    Plants and certain bacteria use a non-mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with specific and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C13 cyclohexenone derivatives and mycorradicin (C14) conjugates, the latter being a major component of the long-known 'yellow pigment'. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identified from mycorrhizal wheat roots after alkaline treatment of an 'apocarotenoid complex' of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the first report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus. PMID:10758508

  8. 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.

  9. Structural organization of the multifunctional animal fatty-acid synthase.

    PubMed

    Witkowski, A; Rangan, V S; Randhawa, Z I; Amy, C M; Smith, S

    1991-06-15

    The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/acetyl transferase, residues 1630-1850 to the enoyl reductase, residues 1870-2100 to the oxyreductase, residues 2114-2190 to the acyl-carrier protein and residues 2200-2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.

  10. 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.

  11. The cellulose synthase superfamily in fully sequenced plants and algae

    PubMed Central

    2009-01-01

    Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ), providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants. PMID:19646250

  12. QSAR modeling of the inhibition of glycogen synthase kinase-3.

    PubMed

    Katritzky, Alan R; Pacureanu, Liliana M; Dobchev, Dimitar A; Fara, Dan C; Duchowicz, Pablo R; Karelson, Mati

    2006-07-15

    Quantitative structure-activity relationship (QSAR) models of the biological activity (pIC50) of 277 inhibitors of Glycogen Synthase Kinase-3 (GSK-3) are developed using geometrical, topological, quantum mechanical, and electronic descriptors calculated by CODESSA PRO. The linear (multilinear regression) and nonlinear (artificial neural network) models obtained link the structures to their reported activity pIC50. The results are discussed in the light of the main factors that influence the inhibitory activity of the GSK-3 enzyme.

  13. Trichodiene synthase: mechanism-based inhibition of a sesquiterpene cyclase.

    PubMed

    Cane, D E; Bowser, T E

    1999-04-19

    The 10-cyclopropylidene analog of farnesyl diphosphate was shown to be a mechanism-based inhibitor of trichodiene synthase with an inactivation rate (k(inact)) of 0.010 +/- 0.0003 min(-1) and an apparent Ki of 663 +/- 75 nM. The presence of three anomalous sesquiterpene products detected in incubation mixtures indicate that the compound also serves as a substrate of the enzyme. PMID:10328298

  14. 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.

  15. 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

  16. [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. PMID:26638491

  17. [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.

  18. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

    PubMed Central

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng

    2016-01-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10–8~10–6 mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10–9 mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase.

  19. 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

  20. Cloning and characterization of a mammalian pseudouridine synthase.

    PubMed Central

    Chen, J; Patton, J R

    1999-01-01

    This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame. PMID:10094309

  1. [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. PMID:26955708

  2. [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.

  3. 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.

  4. 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.

  5. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

    PubMed Central

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng

    2016-01-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10–8~10–6 mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10–9 mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  6. Dynamics of meso and thermo citrate synthases with implicit solvation

    NASA Astrophysics Data System (ADS)

    Cordeiro, J. M. M.

    The dynamics of hydration of meso and thermo citrate synthases has been investigated using the EEF1 methodology implemented with the CHARMM program. The native enzymes are composed of two identical subunits, each divided into a small and large domain. The dynamics behavior of both enzymes at 30°C and 60°C has been compared. The results of simulations show that during the hydration process, each subunit follows a different pathway of hydration, in spite of the identical sequence. The hydrated structures were compared with the crystalline structure, and the root mean square deviation (RMSD) of each residue along the trajectory was calculated. The regions with larger and smaller mobility were identified. In particular, helices belonging to the small domain are more mobile than those of the large domain. In contrast, the residues that constitute the active site show a much lower displacement compared with the crystalline structure. Hydration free energy calculations point out that Thermoplasma acidophilum citrate synthase (TCS) is more stable than chicken citrate synthase (CCS), at high temperatures. Such result has been ascribed to the higher number of superficial charges in the thermophilic homologue, which stabilizes the enzyme, while the mesophilic homologue denatures. These results are in accord with the experimental found that TCS keeps activity at temperatures farther apart from the catalysis regular temperature than the CCS.

  7. The pseudouridine synthases: revisiting a mechanism that seemed settled.

    PubMed

    Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2004-10-13

    RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.

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

    PubMed

    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

  9. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase.

    PubMed

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng; Cui, Jianxiu

    2016-09-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10(-8)~10(-6) mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10(-9) mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  10. 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

  11. 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.

  12. 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.

  13. [Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase].

    PubMed

    Ma, Wenrui; Liu, Chunmei; Yang, Mingfeng; Xue, Feiyan; Chen, Qing; Ma, Lanqing; Lü, Heshu

    2016-02-01

    The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation. PMID:27382775

  14. 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

  15. Energy transduction in the F1 motor of ATP synthase

    NASA Astrophysics Data System (ADS)

    Wang, Hongyun; Oster, George

    1998-11-01

    ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis, after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central `shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

  16. 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

  17. Localization of beta-glucan synthases on the membranes of cultured Lolium multiflorum (ryegrass) endosperm cells.

    PubMed Central

    Henry, R J; Schibeci, A; Stone, B A

    1983-01-01

    The distribution of beta-glucan synthases between plasma membranes and intracellular membranes of suspension-cultured Italian-ryegrass (Lolium multiflorum Lam.) endosperm cells was examined. Highly purified plasma membranes prepared from protoplasts were only slightly enriched in beta-glucan synthases assayed at 10 microM- and 1 mM-UDP-glucose. Most beta-glucan synthase was associated with intracellular membranes. These membranes were fractionated on a linear sucrose density gradient and were resolved into different membrane fractions containing beta-glucan synthases. Beta-Glucan synthases assayed at 10 microM-UDP-glucose were found in a fraction banding at a density of 1.11 g . cm-3, but most of the beta-glucan synthase assayed at 1 mM-DDP-glucose was at a density of 1.04 g . cm-3. PMID:6223621

  18. Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes1[w

    PubMed Central

    Wu, Shuiqin; Schoenbeck, Mark A.; Greenhagen, Bryan T.; Takahashi, Shunji; Lee, Sungbeom; Coates, Robert M.; Chappell, Joseph

    2005-01-01

    A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing β-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for α-barbatene, thujopsene, and β-chamigrene biosynthesis. PMID:15965019

  19. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    PubMed Central

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  20. Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer-nucleotide coacervate droplets.

    PubMed

    Crosby, John; Treadwell, Tom; Hammerton, Michelle; Vasilakis, Konstantinos; Crump, Matthew P; Williams, David S; Mann, Stephen

    2012-12-18

    Compartmentalization of the minimal complex of actinorhodin polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt polyketides under conditions of low and high ionic strength.

  1. 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

  2. The type I fatty acid and polyketide synthases: a tale of two megasynthases.

    PubMed

    Smith, Stuart; Tsai, Shiou-Chuan

    2007-10-01

    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.

  3. Polymorphism and expression of isoflavone synthase genes from soybean cultivars.

    PubMed

    Kim, Hyo-Kyoung; Jang, Yun-Hee; Baek, Il-Sun; Lee, Jeong-Hwan; Park, Min Joo; Chung, Young-Soo; Chung, Jong-Il; Kim, Jeong-Kook

    2005-02-28

    Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings. PMID:15750342

  4. 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

  5. Obesity, insulin resistance, and skeletal muscle nitric oxide synthase

    PubMed Central

    Kraus, Raymond M.; Houmard, Joseph A.; Kraus, William E.; Tanner, Charles J.; Pierce, Joseph R.; Choi, Myung Dong

    2012-01-01

    The molecular mechanisms responsible for impaired insulin action have yet to be fully identified. Rodent models demonstrate a strong relationship between insulin resistance and an elevation in skeletal muscle inducible nitric oxide synthase (iNOS) expression; the purpose of this investigation was to explore this potential relationship in humans. Sedentary men and women were recruited to participate (means ± SE: nonobese, body mass index = 25.5 ± 0.3 kg/m2, n = 13; obese, body mass index = 36.6 ± 0.4 kg/m2, n = 14). Insulin sensitivity was measured using an intravenous glucose tolerance test with the subsequent modeling of an insulin sensitivity index (SI). Skeletal muscle was obtained from the vastus lateralis, and iNOS, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) content were determined by Western blot. SI was significantly lower in the obese compared with the nonobese group (∼43%; P < 0.05), yet skeletal muscle iNOS protein expression was not different between nonobese and obese groups. Skeletal muscle eNOS protein was significantly higher in the nonobese than the obese group, and skeletal muscle nNOS protein tended to be higher (P = 0.054) in the obese compared with the nonobese group. Alternative analysis based on SI (high and low tertile) indicated that the most insulin-resistant group did not have significantly more skeletal muscle iNOS protein than the most insulin-sensitive group. In conclusion, human insulin resistance does not appear to be associated with an elevation in skeletal muscle iNOS protein in middle-aged individuals under fasting conditions. PMID:22797309

  6. 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.

  7. [Nitric oxide and nitric oxide synthase related to male reproduction].

    PubMed

    Ji, Jiajia; Zhao, Yanfang; Chen, Guoyuan

    2007-09-01

    Nitric oxide (NO) may be a kind of signal molecule which may have multiplicate physiological function such as secondary messenger, neurotransmitter and effect molecule. NO may play a crucial role in organism. The production of NO can not get away from nitric oxide synthase (NOS) which may distribute in almost all kind of organs of male reproductive system. NO and NOS may have the function of bifunctional regulation for reproduction. In this paper, the regulatory function of NO and NOS on male reproductive system were reviewed.

  8. 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. PMID:18004775

  9. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

    PubMed Central

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-01

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C22–C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C22–C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer. PMID:18199837

  10. Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

    PubMed

    Culbertson, Justin E; Chung, Dong hee; Ziebart, Kristin T; Espiritu, Eduardo; Toney, Michael D

    2015-04-14

    The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS. PMID:25710100

  11. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

    PubMed

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-22

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

  12. 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.

  13. 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.

  14. 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.

  15. Inhibitors to Polyhydroxyalkanoate (PHA) Synthases: Synthesis, Molecular Docking, and Implications

    PubMed Central

    Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered as an ideal alternative to nonbiodegradable synthetic plastics. However, study of PhaC has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty along with lack of a structure has become the main hurdle to understand and engineer PhaCs for economical PHA production. Here we reported the synthesis of two carbadethia CoA analogs, sT-CH2-CoA 26a and sTet-CH2-CoA 26b as well as sT-aldehyde 29 as new PhaC inhibitors. Study of these analogs with PhaECAv revealed that 26a/b and 29 are competitive and mixed inhibitors, respectively. It was observed that CoA moiety and PHA chain extension can increase binding affinity, which is consistent with the docking study. Estimation from Kic of 26a/b predicts that a CoA analog attached with an octameric-HB chain may facilitate the formation of a kinetically well-behaved synthase. PMID:25394180

  16. 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.

  17. 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.

  18. 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.

  19. GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

    PubMed Central

    Chakravarti, Ritu; Aulak, Kulwant S.; Fox, Paul L.; Stuehr, Dennis J.

    2010-01-01

    Heme proteins play essential roles in biology, but little is known about heme transport inside mammalian cells or how heme is inserted into soluble proteins. We recently found that nitric oxide (NO) blocks cells from inserting heme into several proteins, including cytochrome P450s, hemoglobin, NO synthases, and catalase. This finding led us to explore the basis for NO inhibition and to identify cytosolic proteins that may be involved, using inducible NO synthase (iNOS) as a model target. Surprisingly, we found that GAPDH plays a key role. GAPDH was associated with iNOS in cells. Pure GAPDH bound tightly to heme or to iNOS in an NO-sensitive manner. GAPDH knockdown inhibited heme insertion into iNOS and a GAPDH mutant with defective heme binding acted as a dominant negative inhibitor of iNOS heme insertion. Exposing cells to NO either from a chemical donor or by iNOS induction caused GAPDH to become S-nitrosylated at Cys152. Expressing a GAPDH C152S mutant in cells or providing a drug to selectively block GAPDH S-nitrosylation both made heme insertion into iNOS resistant to the NO inhibition. We propose that GAPDH delivers heme to iNOS through a process that is regulated by its S-nitrosylation. Our findings may uncover a fundamental step in intracellular heme trafficking, and reveal a mechanism whereby NO can govern the process. PMID:20921417

  20. Phytochelatin synthase activity as a marker of metal pollution.

    PubMed

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina; Adam, Vojtech; Zehnalek, Josef; Beklova, Miroslava; Kizek, Rene

    2011-08-30

    The synthesis of phytochelatins is catalyzed by γ-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO(3))(2) for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35°C for 30min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270fkat) in treated cells was more than seven times higher in comparison to control ones. K(m) for PCS was estimated as 2.3mM. PMID:21715087

  1. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    PubMed

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    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.

  2. INHIBITION OF NITRIC OXIDE SYNTHASE BY COBALAMINS AND COBINAMIDES*

    PubMed Central

    Weinberg, J. Brice; Chen, Youwei; Jiang, Ning; Beasley, Bethany E.; Salerno, John C.; Ghosh, Dipak K.

    2009-01-01

    Cobalamins (Cbl) are important co-factors for methionine synthase and methylmalonyl-coA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on 14-C-L-arginine-to-14-C-L-citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide (Cbi), and dicyanocobinamide (CN2-Cbi) potently inhibited all isoforms, whfile cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN2-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN2-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN2-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with L-arginine, tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN2-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles regulating NOS activity in normal and pathological conditions. PMID:19328848

  3. 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.

  4. 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.

  5. 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

  6. Polyketide synthases from poison hemlock (Conium maculatum L.).

    PubMed

    Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

    2015-11-01

    Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs.

  7. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    PubMed Central

    Hargovan, Milan

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors. PMID:24570839

  8. Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages.

    PubMed Central

    Jorens, P. G.; van Overveld, F. J.; Bult, H.; Vermeire, P. A.; Herman, A. G.

    1992-01-01

    1. The synthesis of nitrite and citrulline from L-arginine by immune-stimulated rat alveolar macrophages and the modulation of this synthesis were studied. 2,4-Diamino-6-hydroxypyrimidine (DAHP), 6R-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-sepiapterin were potent inhibitors of the recombinant interferon-gamma induced production of nitrogen oxides in intact cultured cells with I50 values for BH4 and L-sepiapterin of approximately 10 microM. They were equally effective in inhibiting the induced production of citrulline. This inhibitory effect was concentration-dependent for all three modulators investigated. 2. The inhibitory effects were not dependent on incubation times of either 24 or 48 h, on the immune-stimulus used (lipopolysaccharide, interferon-gamma), or whether these stimuli were added during or after the induction period. 3. Pterin-6-carboxylic acid (PCA), which cannot be converted into BH4, and methotrexate (MTX), which inhibits dihydrofolatereductase but not de novo biosynthesis of BH4, did not change the production of nitrite. 4. The data indicate that DAHP, an inhibitor of the de novo biosynthesis of the co-factor BH4, blocks the nitric oxide synthase activity in intact cells. Since the pterins BH4 and L-sepiapterin blocked the L-arginine dependent production of nitrite and citrulline, the activity of nitric oxide synthase in phagocytic cells may be regulated by metabolic endproducts of the de novo biosynthesis of BH4. PMID:1281717

  9. 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.

  10. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    PubMed Central

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-01-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work. PMID:11720289

  11. 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

  12. Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.

    PubMed

    Mühleip, Alexander W; Joos, Friederike; Wigge, Christoph; Frangakis, Achilleas S; Kühlbrandt, Werner; Davies, Karen M

    2016-07-26

    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

  13. Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. 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

  15. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    PubMed

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-11-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

  16. ATP synthase in mycobacteria: special features and implications for a function as drug target.

    PubMed

    Lu, Ping; Lill, Holger; Bald, Dirk

    2014-07-01

    ATP synthase is a ubiquitous enzyme that is largely conserved across the kingdoms of life. This conservation is in accordance with its central role in chemiosmotic energy conversion, a pathway utilized by far by most living cells. On the other hand, in particular pathogenic bacteria whilst employing ATP synthase have to deal with energetically unfavorable conditions such as low oxygen tensions in the human host, e.g. Mycobacterium tuberculosis can survive in human macrophages for an extended time. It is well conceivable that such ATP synthases may carry idiosyncratic features that contribute to efficient ATP production. In this review genetic and biochemical data on mycobacterial ATP synthase are discussed in terms of rotary catalysis, stator composition, and regulation of activity. ATP synthase in mycobacteria is of particular interest as this enzyme has been validated as a target for promising new antibacterial drugs. A deeper understanding of the working of mycobacterial ATP synthase and its atypical features can provide insight in adaptations of bacterial energy metabolism. Moreover, pinpointing and understanding critical differences as compared with human ATP synthase may provide input for the design and development of selective ATP synthase inhibitors as antibacterials. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  17. The action of exogenous abscisic acid on malate-synthase synthesis in germinating castor-bean seeds.

    PubMed

    Dommes, J; Northcote, D H

    1985-12-01

    The presence of 30 μM abscisic acid inhibited development of malate-synthase activity in the endosperm of germinating castor-bean seeds. Malate synthase was purified from castor-bean endosperms and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of malate-synthase mRNA using an in-vitro translation system. The effect of abscisic acid appeared to be greater on malate-synthase mRNA than on the bulk of mRNA, indicating some specificity of abscisic-acid action. The extent of the inhibition of malate-synthase activity and of malate-synthase mRNA accumulation were similar. This indicates that abscisic acid inhibits malate-synthase activity by lowering levels of translatable malate-synthase mRNA rather than by affecting the translation rate of this mRNA.

  18. 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. PMID:26750479

  19. Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA.

    PubMed

    Godzina, S M; Lovato, M A; Meyer, M M; Foster, K A; Wilson, W K; Gu, W; de Hostos, E L; Matsuda, S P

    2000-03-01

    Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

  20. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

    PubMed

    Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

    2015-10-06

    Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

  1. 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

  2. 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

  3. Biochemistry of prostaglandin endoperoxide H synthase-1 and synthase-2 and their differential susceptibility to nonsteroidal anti-inflammatory drugs.

    PubMed

    Smith, W L; DeWitt, D L

    1995-05-01

    The principal pharmacological effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to their ability to inhibit prostaglandin synthesis. NSAIDs block the cyclooxygenase activities of the closely related PGH synthase-1 and PGH synthase-2 (PGHS-1 and PGHS-2) isozymes. NSAIDs are therapeutically useful due to their analgesic, anti-pyretic, anti-inflammatory, and anti-thrombogenic properties. Major side-effects of NSAIDs include their ulcerogenic and nephrotoxic activities. All clinically approved NSAIDs in general use today inhibit both PGHS-1 and PGHS-2. Recently, inhibitors have been identified that are selective toward PGHS-2 and that have potent analgesic and anti-inflammatory activities with minimal ulcerogenic activity. If the new PGHS-2 selective NSAIDs can effectively inhibit inflammatory prostaglandin synthesis by PGHS-2, without inhibiting PGHS-1 prostaglandin synthesis required to regulate sodium and water resorption, and renal blood flow, it is likely that these new drugs will also have significantly less renal toxicity than present-day NSAIDs. In this article, the mechanisms of actions of NSAIDs primarily at the biochemical level, including the reactions catalyzed by PGHSs, will be discussed. In addition, the biochemical properties of these isozymes, and the differential regulation of the PGHS-1 and PGHS-2 genes, will be examined. PMID:7631045

  4. Inhibition of nitric oxide synthase does not impair spatial learning.

    PubMed

    Bannerman, D M; Chapman, P F; Kelly, P A; Butcher, S P; Morris, R G

    1994-12-01

    Nitric oxide (NO), a putative intercellular messenger in the CNS, may be involved in certain forms of synaptic plasticity and learning. This article reports a series of experiments investigating the effects of N omega-nitro-L-arginine methyl ester (L-NAME) upon various forms of learning and memory in the watermaze. L-NAME (75 mg/kg, i.p., sufficient to bring about > 90% inhibition of NO synthesis in brain) produced an apparent impairment in spatial learning when given to naive rats during acquisition (3 d, six training trials per day). This impairment was dose related, stereoselective, and attenuated by coadministration of L-arginine. A second study showed that L-NAME did not affect the retention of a previously learned spatial task. In addition, in a visual discrimination task, the rate at which criterion levels of performance were reached was unaffected by L-NAME. Thus, inhibition of NO synthase may cause a selective impairment of spatial learning without effect upon retention. However, analysis of the early training trials of the visual discrimination task revealed significantly elevated escape latencies in the L-NAME-treated rats, suggesting that inhibition of NO synthase may have more general effects. As normal rats learn the spatial task very rapidly, the possibility arises that the apparent deficit in learning is due to a disruption of some process other than learning per se. A further series of experiments investigated this possibility. L-NAME was found not to impair the learning of a new platform position in the same spatial environment. Surprisingly, L-NAME also had no effect on spatial learning in a second watermaze located in a novel spatial environment by rats well practiced with all aspects of watermaze training. Finally, L-NAME had no effect on spatial learning in naive rats trained with just one trial per day. Thus, systemic injection of an NO synthase inhibitor impairs behavioral performance in two tasks during their initial acquisition, but the

  5. mRNA expressions of inducible nitric oxide synthase, endothelial nitric oxide synthase, and neuronal nitric oxide synthase genes in meningitis patients.

    PubMed

    Oztuzcu, Serdar; Igci, Yusuf Ziya; Arslan, Ahmet; Sivasli, Ercan; Ozkara, Esma; Igci, Mehri; Demiryürek, Seniz; Cengiz, Beyhan; Gogebakan, Bulent; Namiduru, Mustafa; Coskun, Mehmet Yavuz; Cakmak, Ecir Ali

    2011-03-01

    Meningitis is an inflammation of the protective membranes covering the brain and spinal cord caused by bacteria, fungi, or viruses with various clinical symptoms. Although meningitis is not so prevalent, it remains the most serious contagious disease. The aim of our study was to investigate the effect of gene expressions of nitric oxide synthases (NOS) on meningitis patients. Using samples taken from 61 meningitis patients, inducible NOS, endothelial NOS (eNOS), and neuronal NOS mRNA levels were assessed in both blood and cerebrospinal fluid (CSF). A control group was constructed of 64 healthy persons. The gene expression analysis was made using real-time polymerase chain reaction method. There was no neuronal NOS expression in either group, whereas inducible NOS expression was detected in 40 blood samples and 12 CSF samples from meningitis patients. However, there were no marked differences between groups (p=0.5104). eNOS expression was detected in all blood and CSF samples, which was markedly higher in patients (p=0.0367). Because the increase in eNOS expression increases NO production, eNOS expression in meningitis patients is of great importance. This increase of eNOS in meningitis patients compared with healthy subjects may lead to novel treatments for reducing the severity of the disease.

  6. The Interplay between Myc and CTP Synthase in Drosophila

    PubMed Central

    Aughey, Gabriel N.; Grice, Stuart J.; Liu, Ji-Long

    2016-01-01

    CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn. PMID:26889675

  7. Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice

    PubMed Central

    Scherthan, Harry; Horsch, Marion; Beckers, Johannes; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Ford, Steven J.; Burton, Neal C.; Razansky, Daniel; Trümbach, Dietrich; Aichler, Michaela; Walch, Axel Karl; Calzada-Wack, Julia; Neff, Frauke; Wurst, Wolfgang; Hartmann, Tobias; Floss, Thomas

    2016-01-01

    Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility. PMID:27788151

  8. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  9. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering.

    PubMed

    Zerbe, Philipp; Bohlmann, Jörg

    2015-07-01

    Plants produce thousands of diterpenoid natural products; some of which are of significant industrial value as biobased pharmaceuticals (taxol), fragrances (sclareol), food additives (steviosides), and commodity chemicals (diterpene resin acids). In nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. While some diTPSs also form oxygenated compounds, more commonly, oxygenation is achieved by cytochrome P450-dependent mono-oxygenases. Recent genome-, transcriptome-, and metabolome-guided gene discovery and enzyme characterization identified novel diTPS functions that form the core of complex modular pathway systems. Insights into diterpene metabolism may translate into the development of new bioengineered microbial and plant-based production systems.

  10. Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging.

    PubMed

    Pandey, Mukesh K; DeGrado, Timothy R

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

  11. Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase

    SciTech Connect

    Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephan W.; Lee, Richard E.

    2010-01-12

    Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

  12. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    SciTech Connect

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W.

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  13. Farnesyl Diphosphate Synthase Inhibitors With Unique Ligand-Binding Geometries

    PubMed Central

    2015-01-01

    Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis. PMID:25815158

  14. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance.

    PubMed

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara; Benfield, Thomas; Miller, Robert; Rabodonirina, Meja; Helweg-Larsen, Jannik

    2004-10-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about development of resistant organisms. The inability to culture human Pneumocystis, Pneumocystis jirovecii, in a standardized culture system prevents routine susceptibility testing and detection of drug resistance. In other microorganisms, sulfa drug resistance has resulted from specific point mutations in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone.

  15. Catalysis and sulfa drug resistance in dihydropteroate synthase.

    PubMed

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M Brett; Ferreira, Antonio M; Lee, Richard E; Bashford, Donald; White, Stephen W

    2012-03-01

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  16. Sulfa use, dihydropteroate synthase mutations, and Pneumocystis jirovecii pneumonia.

    PubMed

    Stein, Cheryl R; Poole, Charles; Kazanjian, Powel; Meshnick, Steven R

    2004-10-01

    A systematic review was conducted to examine the associations in Pneumocystis jirovecii pneumonia (PCP) patients between dihydropteroate synthase (DHPS) mutations and sulfa or sulfone (sulfa) prophylaxis and between DHPS mutations and sulfa treatment outcome. Selection criteria included study populations composed entirely of PCP patients and mutation or treatment outcome results for all patients, regardless of exposure status. Based on 13 studies, the risk of developing DHPS mutations is higher for PCP patients receiving sulfa prophylaxis than for PCP patients not receiving sulfa prophylaxis (p < 0.001). Results are too heterogeneous (p < 0.001) to warrant a single summary effect estimate. Estimated effects are weaker after 1996 and stronger in studies that included multiple isolates per patient. Five studies examined treatment outcome. The effect of DHPS mutations on treatment outcome has not been well studied, and the few studies that have been conducted are inconsistent even as to the presence or absence of an association.

  17. The Interplay between Myc and CTP Synthase in Drosophila.

    PubMed

    Aughey, Gabriel N; Grice, Stuart J; Liu, Ji-Long

    2016-02-01

    CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn. PMID:26889675

  18. Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging

    PubMed Central

    Pandey, Mukesh K.; DeGrado, Timothy R.

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

  19. Calmodulin-induced structural changes in endothelial nitric oxide synthase

    PubMed Central

    Persechini, Anthony; Tran, Quang-Kim; Black, D.J.; Gogol, Edward P.

    2013-01-01

    We have derived structures of intact calmodulin(CaM)-free and CaM-bound endothelial nitric oxide synthase (eNOS) by reconstruction from cryo-electron micrographs. The CaM-free reconstruction is well fitted by the oxygenase domain dimer, but the reductase domains are not visible, suggesting they are mobile and thus delocalized. Additional protein is visible in the CaM-bound reconstruction, concentrated in volumes near two basic patches on each oxygenase domain. One of these corresponds with a presumptive docking site for the reductase domain FMN-binding module. The other is proposed to correspond with a docking site for CaM. A model is suggested in which CaM binding and docking position the reductase domains near the oxygenase domains and promote docking of the FMN-binding modules required for electron transfer. PMID:23266515

  20. Rotary catalysis of FoF1-ATP synthase.

    PubMed

    Watanabe, Rikiya

    2013-01-01

    The synthesis of ATP, the key reaction of biological energy metabolism, is accomplished by the rotary motor protein; FoF1-ATP synthase (FoF1). In vivo, FoF1, located on the cell membrane, carries out ATP synthesis by using the proton motive force. This heterologous energy conversion is supposed to be mediated by the mechanical rotation of FoF1; however, it still remained unclear. Recently, we developed the novel experimental setup to reproduce the proton motive force in vitro and succeeded in directly observing the proton-driven rotation of FoF1. In this review, we describe the interesting working principles determined so far for FoF1 and then introduce results from our recent study.

  1. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    PubMed

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc. PMID:27017955

  2. New insight into the catalytic properties of rice sucrose synthase.

    PubMed

    Huang, Yu-Chiao; Hsiang, Erh-Chieh; Yang, Chien-Chih; Wang, Ai-Yu

    2016-01-01

    Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.

  3. Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

    PubMed

    Manhas, Reetika; Gowri, Venkatraman Subramanian; Madhubala, Rentala

    2016-01-15

    The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different. PMID:26586914

  4. The Evolution of Function in Strictosidine Synthase-like Proteins

    PubMed Central

    Hicks, Michael A.; Barber, Alan E.; Giddings, Lesley-Ann; Caldwell, Jenna; O’Connor, Sarah E.; Babbitt, Patricia C.

    2013-01-01

    The exponential growth of sequence data provides abundant information for the discovery of new enzyme reactions. Correctly annotating the functions of highly diverse proteins can be difficult, however, hindering use of this information. Global analysis of large superfamilies of related proteins is a powerful strategy for understanding the evolution of reactions by identifying catalytic commonalities and differences in reaction and substrate specificity, even when only a few members have been biochemically or structurally characterized. A comparison of >2500 sequences sharing the six-bladed β-propeller fold establishes sequence, structural and functional links among the three subgroups of the functionally diverse N6P superfamily: the arylesterase-like and senescence marker protein-30/gluconolactonase/luciferin-regenerating enzyme-like (SGL) subgroups, representing enzymes that catalyze lactonase and related hydrolytic reactions, and the so-called “strictosidine synthase-like” (SSL) subgroup. Metal-coordinating residues were identified as broadly conserved in the active sites of all three subgroups except for a few proteins from the SSL subgroup, which have been experimentally determined to catalyze the quite different strictosidine synthase (SS) reaction, a metal-independent condensation reaction. Despite these differences, comparison of conserved catalytic features of the arylesterase-like and SGL enzymes with the SSs identified similar structural and mechanistic attributes between the hydrolytic reactions catalyzed by the former and the condensation reaction catalyzed by SS. The results also suggest that despite their annotations, the great majority of these >500 SSL sequences do not catalyze the SS reaction; rather, they likely catalyze hydrolytic reactions typical of the other two subgroups instead. This prediction was confirmed experimentally for one of these proteins. PMID:21948213

  5. Enzymatic and structural characterization of an archaeal thiamin phosphate synthase.

    PubMed

    Hayashi, Maria; Kobayashi, Kazuya; Esaki, Hiroyoshi; Konno, Hiroyuki; Akaji, Kenichi; Tazuya, Keiko; Yamada, Kazuko; Nakabayashi, Toshikatsu; Nosaka, Kazuto

    2014-04-01

    Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.

  6. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    PubMed

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc.

  7. Identification and characterization of the Populus sucrose synthase gene family.

    PubMed

    An, Xinmin; Chen, Zhong; Wang, Jingcheng; Ye, Meixia; Ji, Lexiang; Wang, Jia; Liao, Weihua; Ma, Huandi

    2014-04-10

    In this study, we indentified 15 sucrose synthase (SS) genes in Populus and the results of RT-qPCR revealed that their expression patterns were constitutive and partially overlapping but diverse. The release of the most recent Populus genomic data in Phytozome v9.1 has revealed the largest SS gene family described to date, comprising 15 distinct members. This information will now enable the analysis of transcript expression profiles for those that have not been previously reported. Here, we performed a comprehensive analysis of SS genes in Populus by describing the gene structure, chromosomal location and phylogenetic relationship of each family member. A total of 15 putative SS gene members were identified in the Populus trichocarpa (Torr. & Gray) genome using the SS domain and amino acid sequences from Arabidopsis thaliana as a probe. A phylogenetic analysis indicated that the 15 members could be classified into four groups that fall into three major categories: dicots, monocots & dicots 1 (M & D 1), and monocots & dicots 2 (M & D 2). In addition, the 15 SS genes were found to be unevenly distributed on seven chromosomes. The two conserved domains (sucrose synthase and glycosyl transferase) were found in this family. Meanwhile, the expression profiles of all 15 gene members in seven different organs were investigated in Populus tomentosa (Carr.) by using RT-qPCR. Additional analysis indicated that the poplar SS gene family is also involved in response to water-deficit. The current study provides basic information that will assist in elucidating the functions of poplar SS family. PMID:24508272

  8. Mechanism and Stereospecificity of a Fully Saturating Polyketide Synthase Module: Nanchangmycin Synthase Module 2 and Its Dehydratase Domain

    PubMed Central

    Guo, Xun; Liu, Tiangang; Valenzano, Chiara R.; Deng, Zixin; Cane, David E.

    2010-01-01

    Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the -SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4-hydroxylpyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed kcat 0.5±0.1 min−1 and Km(1) 19±5 mM at 0.5 mM MM-CoA and kcat(app) 0.26±0.02 min−1 and Km(MM-CoA) 0.11 ±0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(−)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2. PMID:20925339

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

    Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene