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Sample records for 5-phosphate reductoisomerase dxr

  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. 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

  3. Resistance to the Antimicrobial Agent Fosmidomycin and an FR900098 Prodrug through Mutations in the Deoxyxylulose Phosphate Reductoisomerase Gene (dxr)

    PubMed Central

    Armstrong, Christopher M.; Meyers, David J.; Imlay, Leah S.; Freel Meyers, Caren

    2015-01-01

    There is a pressing need for new antimicrobial therapies to combat globally important drug-resistant human pathogens, including Plasmodium falciparum malarial parasites, Mycobacterium tuberculosis, and Gram-negative bacteria, including Escherichia coli. These organisms all possess the essential methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, which is not found in humans. The first dedicated enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr), is inhibited by the phosphonic acid antibiotic fosmidomycin and its analogs, including the N-acetyl analog FR900098 and the phosphoryl analog fosfoxacin. In order to identify mutations in dxr that confer resistance to these drugs, a library of E. coli dxr mutants was screened at lethal fosmidomycin doses. The most resistant allele (with the S222T mutation) alters the fosmidomycin-binding site of Dxr. The expression of this resistant allele increases bacterial resistance to fosmidomycin and other fosmidomycin analogs by 10-fold. These observations confirm that the primary cellular target of fosmidomycin is Dxr. Furthermore, cell lines expressing Dxr-S222T will be a powerful tool to confirm the mechanisms of action of future fosmidomycin analogs. PMID:26124156

  4. [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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes

  19. 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

  20. Novel insights into structure–function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus

    PubMed Central

    Devi, Kamalakshi; Dehury, Budheswar; Phukon, Munmi; Modi, Mahendra Kumar; Sen, Priyabrata

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue-specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future. PMID:25941629

  1. 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

  2. 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

  3. [Cloning and analysis of cDNA encoding key enzyme gene (dxr) of the non-MVA pathway in Taxus chinensis cells].

    PubMed

    Zheng, Qing-Ping; Yu, Long-Jiang; Liu, Zhi; Li, Mo-Yi; Xiang, Fu; Yang, Qin

    2004-07-01

    Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm. PMID:15968987

  4. 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.

  5. Ketol-acid reductoisomerase enzymes and methods of use

    SciTech Connect

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2015-10-27

    Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  6. 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

  7. 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...

  8. 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...

  9. 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...

  10. 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 ...

  11. 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.

  12. 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.

  13. 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

  14. 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…

  15. Crystal Structure Analyses of the Fosmidomycin-Target Enzyme from Plasmodium Falciparum

    NASA Astrophysics Data System (ADS)

    Umeda, Tomonobu; Kusakabe, Yoshio; Tanaka, Nobutada

    The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. Fosmidomycin has proved to be efficient in the treatment of P. falciparum malaria through the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway of isoprenoid biosynthesis, which is absent in humans. Crystal structure analyses of P. falciparum DXR (PfDXR) revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for the induced-fit movement to accommodate the bound inhibitor in the active site, and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We believe that our study will serve as a useful guide to develop more potent PfDXR inhibitors.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Nongenotoxic effects and a reduction of the DXR-induced genotoxic effects of Helianthus annuus Linné (sunflower) seeds revealed by micronucleus assays in mouse bone marrow

    PubMed Central

    2014-01-01

    Background This research evaluated the genotoxicity of oil and tincture of H. annuus L. seeds using the micronucleus assay in bone marrow of mice. The interaction between these preparations and the genotoxic effects of doxorubicin (DXR) was also analysed (antigenotoxicity test). Methods Experimental groups were evaluated at 24-48 h post treatment with N-Nitroso-N-ethylurea (positive control – NEU), DXR (chemotherapeutic), NaCl (negative control), a sunflower tincture (THALS) and two sources of sunflower oils (POHALS and FOHALS). Antigenotoxic assays were carried out using the sunflower tincture and oils separately and in combination with NUE or DXR. Results For THALS, analysis of the MNPCEs showed no significant differences between treatment doses (250–2,000 mg.Kg-1) and NaCl. A significant reduction in MNPCE was observed when THALS (2,000 mg.Kg-1) was administered in combination with DXR (5 mg.Kg-1). For POHALS or FOHALS, analysis of the MNPCEs also showed no significant differences between treatment doses (250–2,000 mg.Kg-1) and NaCl. However, the combination DXR + POHALS (2,000 mg.Kg-1) or DXR + FOHALS (2,000 mg.Kg-1) not contributed to the MNPCEs reduction. Conclusions This research suggests absence of genotoxicity of THALS, dose-, time- and sex-independent, and its combination with DXR can reduce the genotoxic effects of DXR. POHALS and FOHALS also showed absence of genotoxicity, but their association with DXR showed no antigenotoxic effects. PMID:24694203

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

    PubMed

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K B; Snow, Christopher D; Brustad, Eric M; McIntosh, John A; Meinhold, Peter; Zhang, Liang; Arnold, Frances H

    2013-07-01

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  15. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  16. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    SciTech Connect

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  17. Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis.

    PubMed

    Mueller, C; Schwender, J; Zeidler, J; Lichtenthaler, H K

    2000-12-01

    Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

    PubMed

    Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas; Wiig, Jared A; Buller, Andrew R; Einsle, Oliver; Hu, Yilin; Ribbe, Markus W; Arnold, Frances H

    2015-06-15

    Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  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. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Separate and Combined Effects of DNMT and HDAC Inhibitors in Treating Human Multi-Drug Resistant Osteosarcoma HosDXR150 Cell Line

    PubMed Central

    Capobianco, Enrico; Mora, Antonio; La Sala, Dario; Roberti, Annalisa; Zaki, Nazar; Badidi, Elarbi; Taranta, Monia; Cinti, Caterina

    2014-01-01

    Understanding the molecular mechanisms underlying multi-drug resistance (MDR) is one of the major challenges in current cancer research. A phenomenon which is common to both intrinsic and acquired resistance, is the aberrant alteration of gene expression in drug-resistant cancers. Although such dysregulation depends on many possible causes, an epigenetic characterization is considered a main driver. Recent studies have suggested a direct role for epigenetic inactivation of genes in determining tumor chemo-sensitivity. We investigated the effects of the inhibition of DNA methyltransferase (DNMT) and hystone deacethylase (HDAC), considered to reverse the epigenetic aberrations and lead to the re-expression of de novo methylated genes in MDR osteosarcoma (OS) cells. Based on our analysis of the HosDXR150 cell line, we found that in order to reduce cell proliferation, co-treatment of MDR OS cells with DNMT (5-Aza-dC, DAC) and HDAC (Trichostatin A, TSA) inhibitors is more effective than relying on each treatment alone. In re-expressing epigenetically silenced genes induced by treatments, a very specific regulation takes place which suggests that methylation and de-acetylation have occurred either separately or simultaneously to determine MDR OS phenotype. In particular, functional relationships have been reported after measuring differential gene expression, indicating that MDR OS cells acquired growth and survival advantage by simultaneous epigenetic inactivation of both multiple p53-independent apoptotic signals and osteoblast differentiation pathways. Furthermore, co-treatment results more efficient in inducing the re-expression of some main pathways according to the computed enrichment, thus emphasizing its potential towards representing an effective therapeutic option for MDR OS. PMID:24756038

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  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. 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.

  2. A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites

    PubMed Central

    Edwards, Rachel L.; Kelly, Megan L.; Hodge, Dana M.; Tolia, Niraj H.; Odom, Audrey R.

    2014-01-01

    Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. We employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologs in plants and bacteria, other HAD proteins may be MEP pathway regulators. PMID:25058848

  3. 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.

  4. 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

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.

    PubMed

    Bastian, Sabine; Liu, Xiang; Meyerowitz, Joseph T; Snow, Christopher D; Chen, Mike M Y; Arnold, Frances H

    2011-05-01

    2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

  3. Synthesis and biological activities of novel 5-substituted-1,3,4-oxadiazole Mannich bases and bis-Mannich bases as ketol-acid reductoisomerase inhibitors.

    PubMed

    Zhang, Yan; Liu, Xing-Hai; Zhan, Yi-Zhou; Zhang, Li-Yuan; Li, Zheng-Ming; Li, Yong-Hong; Zhang, Xiao; Wang, Bao-Lei

    2016-10-01

    A series of novel 5-substituted-1,3,4-oxadiazole Mannich bases and bis-Mannich bases have been conveniently synthesized in good yields. Their structures were characterized by IR, (1)H NMR, (13)C NMR and elemental analysis. The preliminary bioassay results indicated that some of the compounds showed promising in vitro fungicidal activities towards several test plant fungi; some of them exhibited significant herbicidal activities against Brassica campestris and excellent in vitro inhibitory activities against rice ketol-acid reductoisomerase (KARI). Among 14 novel compounds, 8c, 8d and 8m showed potent KARI inhibitory activities with Ki value of (0.96±0.42), (3.86±0.49) and (3.10±0.71) μmol/L, respectively, and were comparable with IpOHA. These compounds could be novel KARI inhibitors for further investigation. The density functional theory (DFT) calculations and molecular docking were carried out to study the structure-activity relationship (SAR) of the active inhibitors in this Letter. PMID:27575481

  4. 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

  5. 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

  6. 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.

  7. [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

  8. [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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  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. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  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. 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.

  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. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  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. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. [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.

  10. 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

  11. Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

    PubMed

    Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

    2011-10-01

    In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum.

  12. 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.

  13. 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.

  14. A specific process to purify 2-methyl-D-erythritol-4-phosphate enzymatically converted from D-glyceraldehyde-3-phosphate and pyruvate.

    PubMed

    Yang, Shao-Qing; Deng, Jian; Wu, Qian-Qian; Li, Heng; Gao, Wen-Yun

    2015-02-01

    A one-pot enzymatic cascade was established to synthesize MEP, one of the key intermediates in the MEP terpenoid biosynthetic pathway. D-GAP and sodium pyruvate were converted to MEP in a reaction catalyzed by DXP synthase and DXP reductoisomerase (DXR) in the presence of the coenzymes ThPP, NADPH, and Mg2+. The product was then isolated by using a specific two-step purification process and MEP was obtained in a yield of nearly 60% and high purity. Importantly, MEP prepared by this way was totally free from contamination by minor amounts of DXP that was not completely convertible by DXR.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Magnesium and manganese interactively modulate parthenolide accumulation and the antioxidant defense system in the leaves of Tanacetum parthenium.

    PubMed

    Farzadfar, Soudeh; Zarinkamar, Fatemeh; Behmanesh, Mehrdad; Hojati, Mostafa

    2016-09-01

    A balanced nutrient supply is a critical factor affecting accumulation of terpenoids in plants, yet data related to the interactive effects of two essential nutrients for the biosynthesis of sesquiterpenes are scarce. Here, the interactional effects between magnesium (Mg) and manganese (Mn) on plant growth, oxidative status, parthenolide accumulation and expression of key genes involved in parthenolide biosynthesis including 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR), 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR), germacrene A synthase (GAS), germacrene A oxidase (GAO), costunolide synthase (COS) and parthenolide synthase (PTS) in the leaves of feverfew plants grown at different Mn and Mn levels were assessed. Plant growth and leaf pigment concentrations were associated with the amount of applied Mg but could be modified by the Mn level. Deprivation and the addition of both Mg and Mn induce oxidative stress. Mg supply also alleviated the adverse effects of Mn excess on plant growth and oxidative status. In addition, parthenolide biosynthesis decreased under deprivation of Mg or Mn, but the addition of Mn up to 50μM under 2mM Mg supply considerably increased its accumulation. The parthenolide accumulation trend might reflect the up-regulation of terpenoid-related genes and enzyme activities as well as the oxidative status of feverfew leaves. Our data suggest a profound effect of the combined supply of Mg and Mn on parthenolide biosynthesis through the activation of terpene synthases, which concomitantly modulate by oxidative status. PMID:27450490

  4. 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.

  5. Improving the quality of infant sleep through the inclusion at supper of cereals enriched with tryptophan, adenosine-5'-phosphate, and uridine-5'-phosphate.

    PubMed

    Cubero, Javier; Chanclón, Belen; Sánchez, Soledad; Rivero, Montserrat; Rodríguez, Ana Beatriz; Barriga, Carmen

    2009-12-01

    The present study evaluated whether the administration of cereals enriched with nutrients that are facilitators of sleep could help improve the sleep of infants who had sleep disorders at night time. Thirty infants aged 8-16 months with sleep disorders involving at least three nocturnal waking episodes took part in the study. They were given a night-time 'sleep facilitating cereal' product containing 225 mg tryptophan, 5.3 mg adenosine-5'-P, and 6.3 mg uridine-5'-P per 100 g of product. These cereals were given in a double-blind procedure lasting 5 weeks, with ingestion of the cereal between 18:00 and 06:00. In the control week, the children received a standard cereal (75 mg tryptophan/100 g product without nucleotides) dissolved in a standard formula milk (231.5 mg tryptophan, 2.6 mg adenosine-5'-P, 5 mg uridine-5'-P, per 100 g product). In one experimental week, the children received the night-time sleep facilitating cereal together with the standard formula milk. In another week, they received the sleep facilitating cereal together with a night milk specially formulated to attain the sleep rhythm (480 mg tryptophan, 8.8 mg uridine-5'-P, and 7.6 mg adenosine-5'-P per 100 g product). The three experimental weeks were separated by two wash-out weeks in which the milk and cereal administered was identical in composition to that of the control week. All the infants received a programmed writer actimeter which they wore continually, attached to their ankles, to record their motor activity. The recorded activity was used to calculate information about the time in bed, assumed sleep, actual sleep, sleep efficiency, sleep latency, immobility, and total activity. The infants receiving the enriched cereal during the time of darkness showed improvements in their sleep parameters, regardless of whether the milk they took at night was standard or enriched with tryptophan, adenosine-5'-P, and uridine-5'-P. In summary, the administration of enriched cereals led to an improvement in sleep, regardless of the type of infant milk used. These results support the concept of chrononutrition since they confirm that the sleep/wake rhythm can be influenced by diet.

  6. C-H activation in pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate Schiff bases: effect of metal chelation. A computational study.

    PubMed

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

    2013-02-28

    This study reports the carbon acidities of Cα and C4′ atoms in the Schiff bases of pyridoxal-5′-phosphate (PLP) and pyridoxamine-5′-phosphate (PMP) complexed with several biologically available metal ions (Mg2+, Ni2+, Zn2+, Cu2+, Al3+, and Fe3+). Density functional theory calculations were carried out to determine the free energies of proton exchange reactions of a set of 18 carbon acids and a Schiff base used as a reference species. The experimental pK(a) values of such carbon acids were used to calibrate the computed free energies in a range of 30 pK(a) units. Eventually, the pK(a)s of the chelates were obtained by calculating the corresponding free energies against the same reference species and by considering the previous calibration. The carbon acidity of Cα in the chelates of Mg2+, Ni2+, Zn2+, and Cu2+ varies between pK(a) 22 and pK(a) 13 whereas the pK(a) values of C4′ range between 18 and 7. Chelation of trivalent metals Al3+ and Fe3+ causes further decrease of the pK(a) values of Cα and C4′ down to 10 and 5, respectively. The results highlight the efficiency of the combined action of Schiff base formation and metal chelation to activate the Cα carbon of amino acids (pK(a) 29 for zwitterionic alanine). Our results explain that the experimental increase of transamination rates by Zn2+ chelation is due to stabilization of the reactive Schiff base species with respect to the free ligand under physiological pH conditions. However, the increase in reactivity for transamination due to Cu2+ and Al3+ chelation is mostly due to C–H ligand activation. Each metal ion activates the Cα and C4′ carbon atoms to a different extent, which can be exploited to favor specific reactions on the amino acids in aqueous solution. Metal chelation hinders both intramolecular and intermolecular proton-transfer reactions of the imino, phenol, and carboxylate groups. This is the only apparent inconvenience of metal complexes in enzymatic reactions, which, in turn, proposes their consideration for enzyme inhibition. PMID:23387946

  7. The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.

    PubMed

    Cassera, María B; Gozzo, Fabio C; D'Alexandri, Fabio L; Merino, Emilio F; del Portillo, Hernando A; Peres, Valnice J; Almeida, Igor C; Eberlin, Marcos N; Wunderlich, Gerhard; Wiesner, Jochen; Jomaa, Hassan; Kimura, Emilia A; Katzin, Alejandro M

    2004-12-10

    Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

  8. Catalyst-Dependent Syntheses of Phosphatidylinositol-5 Phosphate-DiC8 and its Enantiomer

    PubMed Central

    Kayser-Bricker, Katherine J.; Jordan, Peter A.; Miller, Scott J.

    2008-01-01

    Peptide-based catalysts have been applied to the enantioselective syntheses of the title compounds, with this being the first report of the synthesis of an ent-PI5P analogue. The key steps in the synthesis involve asymmetric phosphorylation catalysis. Additional maneuvers were developed with a protecting groups scheme that enabled efficient, streamlined syntheses of these important mediators of biochemical events. PMID:19606206

  9. Self-assembled DNA crystals: the impact on resolution of 5'-phosphates and the DNA source.

    PubMed

    Sha, Ruojie; Birktoft, Jens J; Nguyen, Nam; Chandrasekaran, Arun Richard; Zheng, Jianping; Zhao, Xinshuai; Mao, Chengde; Seeman, Nadrian C

    2013-02-13

    Designed self-assembled DNA crystals consist of rigid DNA motifs that are held together by cohesive sticky-ended interactions. A prominent application of such systems is that they might be able to act as macromolecular hosts for macromolecular guests, thereby alleviating the crystallization problem of structural biology. We have recently demonstrated that it is indeed possible to design and construct such crystals and to determine their structures by X-ray diffraction procedures. To act as useful hosts that organize biological macromolecules for crystallographic purposes, maximizing the resolution of the crystals will maximize the utility of the approach. The structures reported so far have diffracted only to about 4 Å, so we have examined two factors that might have impact on the resolution. We find no difference in the resolution whether the DNA is synthetic or PCR-generated. However, we find that the presence of a phosphate on the 5'-end of the strands improves the resolution of the crystals markedly.

  10. Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves

    PubMed Central

    Negm, Fayek B.; Marlow, Gary C.

    1985-01-01

    This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H+ → d-ribitol-5-P + NADP+. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. PMID:16664320

  11. Selective inhibition of two soluble adenosine cyclic 3',5'-phosphate phosphodiesterases partially purified from calf liver.

    PubMed

    Yamamoto, T; Lieberman, F; Osborne, J C; Manganiello, V C; Vaughan, M; Hidaka, H

    1984-02-14

    "Low Km" cAMP phosphodiesterase and cGMP-stimulated cyclic nucleotide phosphodiesterase activities were partially purified from calf liver supernatant by chromatography on DEAE-cellulose and DEAE-Sepharose and ammonium sulfate precipitation. The low Km phosphodiesterase was not retained on N6-H2N(CH2)2-cAMP-agarose and could be separated from the cGMP-stimulated phosphodiesterase which was absorbed by this matrix. From the proteins that did not bind, two distinct low Km cAMP phosphodiesterases were separated on Ultrogel AcA 34. One form (fraction C) hydrolyzed cAMP with an apparent Km of approximately 0.5 microM and was very sensitive to inhibition by cGMP. Lineweaver-Burk plots of cAMP hydrolysis by a second form (fraction B) were nonlinear, with an apparent low Km component of approximately 2 microM. This form was rather insensitive to inhibition by cGMP. With both fractions, hydrolysis of cAMP relative to cGMP was much greater at low (approximately 1 microM) than at high (approximately 100 microM) substrate concentrations. Maximal velocities for cAMP and cGMP were similar. From sedimentation equilibrium, the apparent weight-average molecular weight of fraction B was estimated as 174000, and that of fraction C was 85000. Another fraction (A) of cAMP phosphodiesterase eluted at the void volume of the AcA 34 column. On the basis of the relative affinities for cAMP and cGMP and inhibition by cGMP, fraction A is most likely an aggregated form of fraction B. No apparent interconversion of fractions A, B, or C was observed on high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6324851

  12. Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV

    PubMed Central

    Akil, Abdellah; Peng, Juan; Omrane, Mohyeddine; Gondeau, Claire; Desterke, Christophe; Marin, Mickaël; Tronchère, Hélène; Taveneau, Cyntia; Sar, Sokhavuth; Briolotti, Philippe; Benjelloun, Soumaya; Benjouad, Abdelaziz; Maurel, Patrick; Thiers, Valérie; Bressanelli, Stéphane; Samuel, Didier; Bréchot, Christian; Gassama-Diagne, Ama

    2016-01-01

    The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway. PMID:27417143

  13. Plasma Pyridoxal 5'-phosphate (PLP) in the United States population: the National Health and Nutrition Examination Survey, 2003-2004

    Technology Transfer Automated Retrieval System (TEKTRAN)

    No large-scale, population-based study has considered the descriptive epidemiology of vitamin B6 status using the biological marker, plasma pyridoxal 5’ - phosphate (PLP). Consequently, how vitamin B6 status varies with basic demographic and lifestyle factors is unclear. We sought to examine the epi...

  14. Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring.

    PubMed

    Courtois, Martine; Mincheva, Zoia; Andreu, Françoise; Rideau, Marc; Viaud-Massuard, Marie-Claude

    2004-12-01

    Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45-85% at 125 microM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.

  15. Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa.

    PubMed

    Singh, Harsharan; Gahlan, Parul; Kumar, Sanjay

    2013-02-25

    Picrorhiza kurrooa Royle ex Benth. is an economically important medicinal plant known to yield picrosides which have high medicinal value. Picroside I and picroside II are major picrosides associated with various bioactivities. The present work analyzed the expression of various genes of the picrosides biosynthesis pathway in different tissues of the plant in relation to the picrosides content. Eight full-length cDNA sequences namely, 1-deoxy-d-xylulose-5-phosphate synthase (2.317 kb), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (1.767 kb), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (1.674 kb), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (1.701 kb), acetyl-CoA acetyltransferase (1.545 kb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (2.241 kb), isopentenyl pyrophosphate isomerase (987 bp) and geranyl diphosphate synthase (1.434 kb), were cloned to full-length followed by expression analysis of ten genes vis-à-vis picrosides content analysis. There is maximum accumulation of picrosides in leaf tissue followed by the rhizome and root, and a similar pattern of expression was found in all the ten genes. The genes responded to the modulators of the picrosides biosynthesis. Picrosides accumulation was enhanced by application of hydrogen peroxide and abscisic acid, whereas methyl jasmonate and salicylic acid treatment decreased the content.

  16. Role of a Guanidinium Cation-Phosphodianion Pair in Stabilizing the Vinyl Carbanion Intermediate of Orotidine 5'-Phosphate Decarboxylase-Catalyzed Reactions.†

    PubMed Central

    Goryanova, Bogdana; Goldman, Lawrence M.; Amyes, Tina L.; Gerlt, John A; Richard, John P.

    2013-01-01

    The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5'-monophosphate decarboxylase from Saccharomyces cerevisiae (OMPDC) catalyzed reactions of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5'-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d-erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, while the 7.2 kcal/mol side-chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number kex while the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation. PMID:24053466

  17. Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5'-phosphate dependent enzymes L-dopa decarboxylase and alanine racemase.

    PubMed

    Lin, Yen-Lin; Gao, Jiali; Rubinstein, Amir; Major, Dan Thomas

    2011-11-01

    Molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential have been carried out to investigate the internal proton transfer equilibrium of the external aldimine species in l-dopa decarboxylase, and carbanion stabilization by the enzyme cofactor in the active site of alanine racemase. Solvent effects lower the free energy of the O-protonated PLP tautomer both in aqueous solution and in the active site, resulting a free energy difference of about -1 kcal/mol relative to the N-protonated Schiff base in the enzyme. The external aldimine provides the dominant contribution to lowering the free energy barrier for the spontaneous decarboxylation of l-dopa in water, by a remarkable 16 kcal/mol, while the enzyme l-dopa decarboxylase further lowers the barrier by 8 kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary (13)C and the secondary (2)H substitutions. In the case of alanine racemase, if the pyridine ring is unprotonated as that in the active site, there is destabilizing contribution to the formation of the α-carbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the α-carbanion is stabilized both when the pyridine ring is protonated and unprotonated. The computational studies illustrated in this article show that combined QM/MM simulations can help provide a deeper understanding of the mechanisms of PLP-dependent enzymes. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

  18. 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.

  19. Kinetics of the template-directed oligomerization of guanosine 5'-phosphate-2-methylimidazolide: Effect of temperature on individual steps of reactionion

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Bernasconi, C. F.; Alberas, D. J.

    1991-01-01

    Non-enzymatic, template-directed reactions have been proposed as models for prebiological polynucleotide synthesis. Chemically activated mononucleotides react in the presence of a polynucleotide, acting as the template in a Watson-Crick base-pairing fashing, and form the complementary daughter polynucleotide. Phosphoimidazolide-activated nucleotides have been used successfully as substrates in these reactions. The kinetics of the guanosine 5'-monophosphate-2-methylimidazolide (2-MelmpG) reaction in aqueous pH 8.0 solutions in the presence and in the absence of polycytidylate (poly(C)) were studied, acting as the template at 6, 23, and 37 C. In the absence of the template, the major reaction pathway of 2-MelmpG is hydrolysis of the P-N bond to form the unreactive guanosine 5'-monophosphate (5'-GMP) and 2-methylimidazole. Concentrated solution of 2-MelmpG (greater than 0.02 M) in the absence of the template form only a small amount dinucleotide, (pG)2, but in the presence of poly(C), oligoguanylates, (pG)n with 2 less than or = n less than or = 40, can be detected. We were able to determine the rate constants for individual steps of this reaction. A summary of the conclusions is presented.

  20. Biochemical assessments of retinol, alpha-tocopherol, pyridoxal--5-phosphate oxidative stress index and total antioxidant status in adolescent professional basketball players and sedentary controls.

    PubMed

    Yilmaz, Necat; Erel, Ozcan; Hazer, Muhsin; Bağci, Cahit; Namiduru, Emine; Gül, Ece

    2007-01-01

    Physical training is known to increase the antioxidant defence system and reduce exercise-induced oxidative stress. However, intense physical aerobic and anaerobic training with competition, such as those imposed on young professional basketball players can induce an increase of oxidative stress, which can be implicated with overtraining. The aim of this study was to test the effect of training and competition load on oxidative stress, antioxidant status, and vitamin levels in basketball players. Oxidative Stres Index (OSI 1), Total Peroxide (TPx) antioxidant (vitamin E, A and The total antioxidant status (TAC 1)), biochemical lipid parameters, as well as training results were measured. Results showed that all plasma vitamin levels were significantly higher in basketball players (vitamin A: 1.61 +/- 0.05 mmol/l, vitamin E: 26.45 +/- 0.72 mmol/l, vitamin B6: 10.58 +/- 0.7 mgr/l) than sedentary controls (vitamin A: 1.22 +/- 0.04 mmol /l, vitamin E: 19.24 +/- 0.73 mmol/l, vitamin B6: 6.0 +/- 0.35 mgr/l) (p < 0.01). In addition TAC 1 was 2.06 +/- 0.02 and 1.89 +/- 0.01 mmol Trolox eq/L in basketball players and controls, respectively (p < 0.01). Conversely OSI was 0.89 +/- 0.09 arbitrary unit and 0.88 +/- 0.071 arbitrary unit in basketball players and controls, respectively (p > 0.05). However, total plasma peroxide level (TPx) of basketball players and controls was not statistically different (18.55 +/- 2.07 and 17.18 +/- 1.61 micromol H2O2/L, respectively; p > 0.05). We conclude that physical exercise increase antioxidant levels and cause balance of the homeostasis. Training can not have positive or negative effects on oxidative stress depending on training load. The results suggested that oxidative stress and antioxidant measurement are significant in the biological follow-up of young basketball players.

  1. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes.

    PubMed

    Mandal, Shantanu; Upadhyay, Shivangi; Singh, Ved Pal; Kapoor, Rupam

    2015-04-01

    Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants. PMID:25734328

  2. Dexrazoxane Diminishes Doxorubicin-Induced Acute Ovarian Damage and Preserves Ovarian Function and Fecundity in Mice

    PubMed Central

    Ringelstetter, Ashley; Khatib, Hasan; Abbott, David H.; Salih, Sana M.

    2015-01-01

    Advances in cancer treatment utilizing multiple chemotherapies have dramatically increased cancer survivorship. Female cancer survivors treated with doxorubicin (DXR) chemotherapy often suffer from an acute impairment of ovarian function, which can persist as long-term, permanent ovarian insufficiency. Dexrazoxane (Dexra) pretreatment reduces DXR-induced insult in the heart, and protects in vitro cultured murine and non-human primate ovaries, demonstrating a drug-based shield to prevent DXR insult. The present study tested the ability of Dexra pretreatment to mitigate acute DXR chemotherapy ovarian toxicity in mice through the first 24 hours post-treatment, and improve subsequent long-term fertility throughout the reproductive lifespan. Adolescent CD-1 mice were treated with Dexra 1 hour prior to DXR treatment in a 1:1 mg or 10:1 mg Dexra:DXR ratio. During the acute injury period (2–24 hours post-injection), Dexra pretreatment at a 1:1 mg ratio decreased the extent of double strand DNA breaks, diminished γH2FAX activation, and reduced subsequent follicular cellular demise caused by DXR. In fertility and fecundity studies, dams pretreated with either Dexra:DXR dose ratio exhibited litter sizes larger than DXR-treated dams, and mice treated with a 1:1 mg Dexra:DXR ratio delivered pups with birth weights greater than DXR-treated females. While DXR significantly increased the “infertility index” (quantifying the percentage of dams failing to achieve pregnancy) through 6 gestations following treatment, Dexra pretreatment significantly reduced the infertility index following DXR treatment, improving fecundity. Low dose Dexra not only protected the ovaries, but also bestowed a considerable survival advantage following exposure to DXR chemotherapy. Mouse survivorship increased from 25% post-DXR treatment to over 80% with Dexra pretreatment. These data demonstrate that Dexra provides acute ovarian protection from DXR toxicity, improving reproductive health in a mouse

  3. Sustained Delivery of a HIF-1 Antagonist for Ocular Neovascularization

    PubMed Central

    Iwase, Takeshi; Fu, Jie; Yoshida, Tsunehiko; Muramatsu, Daisuke; Miki, Akiko; Hashida, Noriyasu; Lu, Lili; Oveson, Brian; Silva, Raquel Lima e; Seidel, Christopher; Yang, Ming; Connelly, Sheila; Shen, Jikui; Han, Bing; Wu, Mingsheng; Semenza, Gregg L.; Hanes, Justin; Campochiaro, Peter A.

    2013-01-01

    Doxorubicin (DXR) and daunorubicin (DNR) inhibit hypoxia-inducible factor-1 (HIF-1) transcriptional activity by blocking its binding to DNA. Intraocular injections of DXR or DNR suppressed choroidal and retinal neovascularization (NV), but also perturbed retinal function as demonstrated by electroretinograms (ERGs). DXR was conjugated to novel copolymers of branched polyethylene glycol and poly(sebacic acid) (DXR-PSA-PEG3) and formulated into nanoparticles that when placed in aqueous buffer, slowly released small DXR-conjugates. Intraocular injection of DXR-PSA-PEG3 nanoparticles (1 or 10 μg DXR content) reduced HIF-1-responsive gene products, strongly suppressed choroidal and retinal NV, and did not cause retinal toxicity. In transgenic mice that express VEGF in photoreceptors, intraocular injection of DXR-PSA-PEG3 nanoparticles (10 μg DXR content) suppressed NV for at least 35 days. Intraocular injection of DXR-PSA-PEG3 nanoparticles (2.7 mg DXR content) in rabbits resulted in sustained DXR-conjugate release with detectable levels in aqueous humor and vitreous for at least 105 days. This study demonstrates a novel HIF-1-inhibitor-polymer conjugate formulated into controlled-release particles that maximizes efficacy and duration of activity, minimizes toxicity, and provides a promising new chemical entity for treatment of ocular NV. PMID:24126220

  4. Sustained delivery of a HIF-1 antagonist for ocular neovascularization.

    PubMed

    Iwase, Takeshi; Fu, Jie; Yoshida, Tsunehiko; Muramatsu, Daisuke; Miki, Akiko; Hashida, Noriyasu; Lu, Lili; Oveson, Brian; Lima e Silva, Raquel; Seidel, Christopher; Yang, Ming; Connelly, Sheila; Shen, Jikui; Han, Bing; Wu, Mingsheng; Semenza, Gregg L; Hanes, Justin; Campochiaro, Peter A

    2013-12-28

    Doxorubicin (DXR) and daunorubicin (DNR) inhibit hypoxia-inducible factor-1 (HIF-1) transcriptional activity by blocking its binding to DNA. Intraocular injections of DXR or DNR suppressed choroidal and retinal neovascularization (NV), but also perturbed retinal function as demonstrated by electroretinograms (ERGs). DXR was conjugated to novel copolymers of branched polyethylene glycol and poly(sebacic acid) (DXR-PSA-PEG3) and formulated into nanoparticles that when placed in aqueous buffer, slowly released small DXR-conjugates. Intraocular injection of DXR-PSA-PEG3 nanoparticles (1 or 10 μg DXR content) reduced HIF-1-responsive gene products, strongly suppressed choroidal and retinal NV, and did not cause retinal toxicity. In transgenic mice that express VEGF in photoreceptors, intraocular injection of DXR-PSA-PEG3 nanoparticles (10 μg DXR content) suppressed NV for at least 35 days. Intraocular injection of DXR-PSA-PEG3 nanoparticles (2.7 mg DXR content) in rabbits resulted in sustained DXR-conjugate release with detectable levels in aqueous humor and vitreous for at least 105 days. This study demonstrates a novel HIF-1-inhibitor-polymer conjugate formulated into controlled-release particles that maximizes efficacy and duration of activity, minimizes toxicity, and provides a promising new chemical entity for treatment of ocular NV. PMID:24126220

  5. Improving peppermint essential oil yield and composition by metabolic engineering.

    PubMed

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R; Turner, Glenn W; Davis, Edward M; Lange, Iris; Baker, Raymond C; Boydston, Rick A; Croteau, Rodney B

    2011-10-11

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost. PMID:21963983

  6. Improving peppermint essential oil yield and composition by metabolic engineering.

    PubMed

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R; Turner, Glenn W; Davis, Edward M; Lange, Iris; Baker, Raymond C; Boydston, Rick A; Croteau, Rodney B

    2011-10-11

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

  7. Proteomics analysis of UV-irradiated Lonicera japonica Thunb. with bioactive metabolites enhancement.

    PubMed

    Zhang, Lin; Li, Ximin; Zheng, Wen; Fu, Zhirong; Li, Wenting; Ma, Luyu; Li, Ke; Sun, Lianli; Tian, Jingkui

    2013-12-01

    A previous study showed that the contents of caffeoylquinic acids and iridoids, the major bioactive components in the postharvest Lonicera japonica Thunb., were induced by enhanced ultraviolet (UV)-A or UV-B irradiation. To clarify the UV-responsive key enzymes in the bioactive metabolites biosynthetic pathway and the related plant defense mechanism in L. japonica, 2DE in combination with MALDI-TOF/TOF MS was employed. Seventy-five out of 196 differential proteins were positively identified. Based on the functions, these proteins were grouped into nine categories, covering a wide range of molecular processes including the secondary metabolites (caffeoylquinic acids and iridoids) biosynthetic-related proteins, photosynthesis, carbohydrate and energy metabolism, stress, DNA, transport-related proteins, lipid metabolism, amino acid metabolism, cell wall. Of note is the increasing expression of 1-deoxy-d-xylulose 5-phosphate reductoisomerase and 5-enol-pyruvylshikimate-phosphate synthase, which was crucial to supply more precursor for the secondary metabolites including caffeoylquinic acids and iridoids. Thus, this study provides both the clues at the protein level for the increase of the two bioactive components upon UV irradiation and the profile of UV-responsive proteins in L. japonica.

  8. Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

    PubMed

    Hemmerlin, Andréa; Hoeffler, Jean-François; Meyer, Odile; Tritsch, Denis; Kagan, Isabelle A; Grosdemange-Billiard, Catherine; Rohmer, Michel; Bach, Thomas J

    2003-07-18

    In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

  9. Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis.

    PubMed

    Sando, Tomoki; Takeno, Shinya; Watanabe, Norie; Okumoto, Hiroshi; Kuzuyama, Tomohisa; Yamashita, Atsushi; Hattori, Masahira; Ogasawara, Naotake; Fukusaki, Eiichiro; Kobayashi, Akio

    2008-11-01

    Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

  10. FR-900098, an antimalarial development candidate that inhibits the non-mevalonate isoprenoid biosynthesis pathway, shows no evidence of acute toxicity and genotoxicity

    PubMed Central

    Wiesner, Jochen; Ziemann, Christina; Hintz, Martin; Reichenberg, Armin; Ortmann, Regina; Schlitzer, Martin; Fuhst, Rainer; Timmesfeld, Nina; Vilcinskas, Andreas; Jomaa, Hassan

    2016-01-01

    ABSTRACT FR-900098 is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase, the second enzyme in the non-mevalonate isoprenoid biosynthesis pathway. In previous studies, FR-900098 was shown to possess potent antimalarial activity in vitro and in a murine malaria model. In order to provide a basis for further preclinical and clinical development, we studied the acute toxicity and genotoxicity of FR-900098. We observed no acute toxicity in rats, i.e. there were no clinical signs of toxicity and no substance-related deaths after the administration of a single dose of 3000 mg/kg body weight orally or 400 mg/kg body weight intravenously. No mutagenic potential was detected in the Salmonella typhimurium reverse mutation assay (Ames test) or an in vitro mammalian cell gene mutation test using mouse lymphoma L5178Y/TK+/− cells (clone 3.7.2C), both with and without metabolic activation. In addition, FR-900098 demonstrated no clastogenic or aneugenic capability or significant adverse effects on blood formation in an in vivo micronucleus test with bone marrow erythrocytes from NMRI mice. We conclude that FR-900098 lacks acute toxicity and genotoxicity, supporting its further development as an antimalarial drug. PMID:27260413

  11. Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys.

    PubMed Central

    Peeters, C C; Evenberg, D; Hoogerhout, P; Käyhty, H; Saarinen, L; van Boeckel, C A; van der Marel, G A; van Boom, J H; Poolman, J T

    1992-01-01

    Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections. PMID:1563770

  12. Dexrazoxane exacerbates doxorubicin-induced testicular toxicity.

    PubMed

    Levi, Mattan; Tzabari, Moran; Savion, Naphtali; Stemmer, Salomon M; Shalgi, Ruth; Ben-Aharon, Irit

    2015-10-01

    Infertility induced by anti-cancer treatments pose a major concern for cancer survivors. Doxorubicin (DXR) has been previously shown to exert toxic effects on the testicular germinal epithelium. Based upon the cardioprotective traits of dexrazoxane (DEX), we studied its potential effect in reducing DXR-induced testicular toxicity. Male mice were injected with 5  mg/kg DXR, 100  mg/kg DEX, combination of both or saline (control) and sacrificed either 1, 3 or 6 months later. Testes were excised and further processed. Glutathione and apoptosis assays were performed to determine oxidative stress. Immunohistochemistry and confocal microscopy were used to study the effects of the drugs on testicular histology and on spermatogonial reserve. DXR and the combined treatment induced a striking decline in testicular weight. DEX prevented DXR-induced oxidative stress, but enhanced DXR-induced apoptosis within the testes. Furthermore, the combined treatment depleted the spermatogonial reserve after 1 month, with impaired recovery at 3 and 6 months post-treatment. This resulted in compromised sperm parameters, testicular and epididymal weights as well as significantly reduced sperm motility, all of which were more severe than those observed in DXR-treated mice. The activity of DEX in the testis may differ from its activity in cardiomyocytes. Adding DEX to DXR exacerbates DXR-induced testicular toxicity. PMID:26329125

  13. Response and Defense Mechanisms of Taxus chinensis Leaves Under UV-A Radiation are Revealed Using Comparative Proteomics and Metabolomics Analyses.

    PubMed

    Zheng, Wen; Komatsu, Setsuko; Zhu, Wei; Zhang, Lin; Li, Ximin; Cui, Lei; Tian, Jingkui

    2016-09-01

    Taxus chinensis var. mairei is a species endemic to south-eastern China and one of the natural sources for the anticancer medicine paclitaxel. To investigate the molecular response and defense mechanisms of T. chinensis leaves to enhanced ultraviolet-A (UV-A) radiation, gel-free/label-free and gel-based proteomics and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The transmission electron microscopy results indicated damage to the chloroplast under UV-A radiation. Proteomics analyses in leaves and chloroplasts showed that photosynthesis-, glycolysis-, secondary metabolism-, stress-, and protein synthesis-, degradation- and activation-related systems were mainly changed under UV-A radiation. Forty-seven PSII proteins and six PSI proteins were identified as being changed in leaves and chloroplasts under UV-A treatment. This indicated that PSII was more sensitive to UV-A than PSI as the target of UV-A light. Enhanced glycolysis, with four glycolysis-related key enzymes increased, provided precursors for secondary metabolism. The 1-deoxy-d-xylulose-5-phosphate reductoisomerase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase were identified as being significantly increased during UV-A radiation, which resulted in paclitaxel enhancement. Additionally, mRNA expression levels of genes involved in the paclitaxel biosynthetic pathway indicated a down-regulation under UV-A irradiation and up-regulation in dark incubation. These results reveal that a short-term high dose of UV-A radiation could stimulate the plant stress defense system and paclitaxel production. PMID:27318281

  14. Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

    PubMed

    Rasulov, Bahtijor; Talts, Eero; Kännaste, Astrid; Niinemets, Ülo

    2015-06-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

  15. A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus.

    PubMed

    Matsuzaki, Motomichi; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Kita, Kiyoshi; Nozaki, Hisayoshi

    2008-06-01

    Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event.

  16. Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

    PubMed Central

    2015-01-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

  17. Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L

    PubMed Central

    2011-01-01

    Background Recently, Artemisia annua L. (annual or sweet wormwood) has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues. Results The four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13) reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures). Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves. Conclusions Four genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on

  18. Dexrazoxane Abrogates Acute Doxorubicin Toxicity in Marmoset Ovary1

    PubMed Central

    Salih, Sana M.; Ringelstetter, Ashley K.; Elsarrag, Mazin Z.; Abbott, David H.; Roti, Elon C. Roti

    2015-01-01

    ABSTRACT Preservation of ovarian function following chemotherapy for nonovarian cancers is a formidable challenge. For prepubescent girls, the only option to prevent chemotherapy damage to the ovary is ovarian tissue cryopreservation, an experimental procedure requiring invasive surgeries to harvest and reimplant tissue, which carries the risk of cancer reintroduction. Drugs that block the primary mechanism of chemotherapy insult, such as dexrazoxane (Dexra) in the context of anthracycline chemotherapy, provide a novel approach for ovarian protection and have the potential to overcome current limitations to oncofertility treatment. Dexra is a catalytic topoisomerase 2 inhibitor that protects the mouse ovary from acute doxorubicin (DXR) chemotherapy toxicity in vitro by preventing DXR-induced DNA damage and subsequent gammaH2AX activation. To translate acute DXR ovarian insult and Dexra protection from mouse to nonhuman primate, freshly obtained marmoset ovarian tissue was cultured in vitro and treated with vehicle or 20 μM Dexra 1 h prior to 50 nM DXR. Cultured ovarian tissue was harvested at 2, 4, or 24 h post-DXR treatment. Dexra prevented DXR-induced DNA double-strand breaks as quantified by the neutral comet assay. DXR treatment for 24 h increased gammaH2AX phosphorylation, specifically increasing the number of foci-positive granulosa cells in antral follicles, while Dexra pretreatment inhibited DXR-induced gammaH2AX phosphorylation foci formation. Additionally, Dexra pretreatment trended toward attenuating DXR-induced AKT1 phosphorylation and caspase-9 activation as assayed by Western blots of ovarian tissue lysates. The combined findings suggest Dexra prevents primary DXR-induced DNA damage, the subsequent cellular response to DNA damage, and may diminish early apoptotic signaling in marmoset ovarian tissue. This study provides initial translation of Dexra protection against acute ovarian DXR toxicity from mice to marmoset monkey tissue. PMID:25609833

  19. Protective effect of CardiPro against doxorubicin-induced cardiotoxicity in mice.

    PubMed

    Mohan, I K; Kumar, K V; Naidu, M U R; Khan, M; Sundaram, C

    2006-03-01

    The effect of CardiPro, a polyherbal formulation, with an antioxidant property, has been studied on doxorubicin (DXR)-induced cardiotoxicity in mice. CardiPro (150 mg/kg b.w., twice daily was administered orally for 7 weeks along with four equal injections (each containing 4.0 mg/kg b.w., DXR) intraperitoneally, once weekly (cumulative dose 16 mg/kg). After a 3-week post DXR treatment period, cardiotoxicity was assessed by noting mortality, volume of ascites, liver congestion, changes in heart weight, myocardial lipid peroxidation, antioxidant enzymes and histology of heart. DXR-treated animals showed higher mortality (50%) and more ascites. Myocardial SOD and glutathione peroxidase activity were decreased and lipid peroxidation was increased. Histology of heart of DXR-treated animals showed loss of myofibrils and focal cytoplasmic vacuolization. CardiPro significantly protected the mice from DXR-induced cardiotoxic effects as evidenced by lower mortality (25%), less ascites, myocardial lipid peroxidation, normalization of antioxidant enzymes and minimal damage to the heart histologically. Our data confirm the earlier reports that DXR cardiotoxicity is associated with the free radical-induced tissue damage. Administration of CardiPro, with an antioxidant property, protected the DXR-induced cardiotoxicity in mice. PMID:16492523

  20. The carotenogenesis pathway via the isoprenoid-beta-carotene interference approach in a new strain of Dunaliella salina isolated from Baja California Mexico.

    PubMed

    Paniagua-Michel, J; Capa-Robles, Willian; Olmos-Soto, Jorge; Gutierrez-Millan, Luis Enrique

    2009-01-01

    D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.

  1. Bortezomib Prevents Acute Doxorubicin Ovarian Insult and Follicle Demise, Improving the Fertility Window and Pup Birth Weight in Mice

    PubMed Central

    Roti Roti, Elon C.; Ringelstetter, Ashley K.; Kropp, Jenna; Abbott, David H.; Salih, Sana M.

    2014-01-01

    Increasing numbers of female patients survive cancer, but succumb to primary ovarian insufficiency after chemotherapy. We tested the hypothesis that Bortezomib (Bort) protects ovaries from doxorubicin (DXR) chemotherapy by treating female mice with Bort 1 hour prior to DXR. By preventing DXR accumulation in the ovary, Bort attenuated DXR-induced DNA damage in all ovarian cell types, subsequent γH2AFX phosphorylation, and resulting apoptosis in preantral follicles. Bort pretreatment extended the number of litters per mouse, improved litter size and increased pup weight following DXR treatment, thus increasing the duration of post-chemotherapy fertility and improving pup health. As a promising prophylactic ovoprotective agent, Bort does not interfere with cancer treatment, and is currently used as a chemotherapy adjuvant. Bort-based chemoprotection may preserve ovarian function in a non-invasive manner that avoids surgical ovarian preservation, thus diminishing the health complications of premature menopause following cancer treatment. PMID:25251158

  2. Characteristics of tissue distribution of various polysaccharides as drug carriers: influences of molecular weight and anionic charge on tumor targeting.

    PubMed

    Sugahara, S; Okuno, S; Yano, T; Hamana, H; Inoue, K

    2001-05-01

    Using the Walker 256 model for carcinosarcoma-bearing rats, we intravenously administered 5 polysaccharide carriers with various molecular weights (MWs) and electric charges and tested for their plasma and tissue distribution. Two carriers, carboxymethylated-D-manno-D-glucan (CMMG) and CMdextran (CMDex), showed higher plasma AUC than the other carriers tested, namely, CMchitin (CMCh), N-desulfated N-acetylated heparin (DSH), and hyaluronic acid (HA). This was consistently found to be true over the range of MWs tested. For CMDex, the maximum value of plasma AUC was obtained when the MW exceeded 150 kDa. As for the anionic charge, CMDex (110-180 kDa) with a degree of substitution (DS) of the CM groups ranging from 0.2 to 0.6, showed maximum plasma AUC values. Twenty-four hours after administration, the concentration of CMDex (180-250 kDa; DS: 0.6-1.2) in tumors was more than 3% of dose/g--approximately 10-fold higher than those observed with CMCh, DSH and HA. Doxorubicin (DXR) was bound to these carriers via a peptide spacer, GlyGlyPheGly (GGFG), to give carrier-GGFG-DXR conjugates (DXR content: 4.2-7.0 (w/w)%), and the antitumor effects of these conjugates were tested with Walker 256 carcinosarcoma-bearing rats by monitoring the tumor weights after a single intravenous injection. Compared with free DXR, CMDex-GGFG-DXR and CMMG-GGFG-DXR conjugates significantly suppressed tumor growth, while the CMCh-GGFG-DXR, DSH-GGFG-DXR, and HA-GGFG-DXR conjugates in a similar comparison showed weak tumor growth inhibition. These findings suggest that the antitumor effect of the carrier-DXR conjugates was related to the extent with which the carriers accumulated in the tumors.

  3. Reference values for digital X-ray radiogrammetry parameters in children and adolescents in comparison to estimates in patients with distal radius fractures.

    PubMed

    Renz, Diane M; Malich, Ansgar; Ulrich, Andreas; Pfeil, Alexander; Mentzel, Hans-Joachim; Streitparth, Florian; Maurer, Martin H; Teichgräber, Ulf K; Böttcher, Joachim

    2016-01-01

    The first objective of this study was to determine normative digital X-ray radiogrammetry (DXR) values, based on original digital images, in a pediatric population (aged 6-18 years). The second aim was to compare these reference data with patients suffering from distal radius fractures, whereas both cohorts originated from the same geographical region and were evaluated using the same technical parameters as well as inclusion and exclusion criteria. DXR-BMD and DXR-MCI of the metacarpal bones II-IV were assessed on standardized digital hand radiographs, without printing or scanning procedures. DXR parameters were estimated separately by gender and among six age groups; values in the fracture group were compared to age- and gender-matched normative data using Student's t tests and Z scores. In the reference cohort (150 boys, 138 girls), gender differences were found in bone mineral density (DXR-BMD), with higher values for girls from 11 to 14 years and for boys from 15 to 18 years (p < 0.05). Girls had higher normative metacarpal index (DXR-MCI) values than boys, with significant differences at 11-14 years (p < 0.05). In the case-control investigation, the fracture group (95 boys, 69 girls) presented lower DXR-BMD at 15-18 years in boys and 13-16 years in girls vs. the reference cohort (p < 0.05); DXR-MCI was lower at 11-18 years in boys and 11-16 years in girls (p < 0.05). Mean Z scores in the fracture group for DXR-BMD were -0.42 (boys) and -0.46 (girls), and for DXR-MCI were -0.51 (boys) and -0.53 (girls). These findings indicate that the fully digital DXR technique can be accurately applied in pediatric populations ≥ 6 years of age. The lower DXR-BMD and DXR-MCI values in the fracture group suggest promising early identification of individuals with increased fracture risk, without the need for additional radiation exposure, enabling the initiation of prevention strategies to possibly reduce the incidence of osteoporosis later in life.

  4. Metacarpal Index Estimated by Digital X-ray Radiogrammetry as a Tool for Differentiating Rheumatoid Arthritis Related Periarticular Osteopenia

    PubMed Central

    Böttcher, Joachim; Pfeil, Alexander; Petrovitch, Alexander; Schmidt, Mirco; Kramer, Anika; Schäfer, Max Ludwig; Gajda, Mieczyslaw; Hein, Gert; Wolf, Gunter; Kaiser, Werner A.

    2006-01-01

    To investigate Metacarpal Index (MCI) and Bone Mineral Density (BMD) estimated by Digital X-ray Radiogrammetry (DXR) with respect to its ability to quantify severity-dependent variations of bone mineralisation in patients with early rheumatoid arthritis compared to Dual Energy X-ray Absorptiometry (DXA), 122 patients underwent a prospective analysis of BMD and MCI by DXR, whereas both DXR-parameters were estimated from plain radiographs of the non-dominant hand. In comparison DXA measured BMD on total femur and lumbar spine (L2-L4). Additionally Steinbrocker Stage was assessed to differentiate the severity of rheumatoid arthritis (RA). Disease activity of RA was estimated by C-reactive Protein (CRP; in mg/l), Erythrocyte Sedimentation Rate (ESR in mm/1st hour) and by the disease activity score with 28-joint count (DAS 28). In consequence, The DXR-parameters, in particular DXR-MCI, revealed significant associations to age, Body Mass Index, CRP, DAS 28 and Steinbrocker graduation; no significant associations could be verified between DXA-parameters and all characteristics of disease activity and severity of RA. The highest correlation was found between DXR-MCI and DXR-BMD with R=0.89 (independent from severity of RA). In all patients DXR-MCI significantly decreased (-14.3%) from 0.42 ± 0.09 (stage 1) to 0.36 ± 0.07 (stage 2) dependent on severity of RA. The comparable relative reduction of DXR-BMD was -11.1%. The group of patients with minor disease activity (DAS 28>5.1) showed a significant flattened reduction (-11.4%) for DXR-MCI from 0.44 ± 0.08 (stage 1) to 0.39 ± 0.08 (stage 2). For accentuated disease activity (DAS 28>5.1) the DXR-MCI revealed a pronounced reduction (-23.1 %). No significant declines were observed for DXA-BMD of the lumbar spine and total femur in all patients as well as dependent on disease activity. Conclusion: DXR can exactly quantify cortical thinning of the metacarpal bones and can identify cortical demineralisation in patients

  5. Sonosensitive dioleoylphosphatidylethanolamine-containing liposomes with prolonged blood circulation time of doxorubicin.

    PubMed

    Evjen, Tove J; Hagtvet, Eirik; Nilssen, Esben A; Brandl, Martin; Fossheim, Sigrid L

    2011-07-17

    Ultrasound sensitive (sonosensitive) liposomes represent a drug delivery system designed for releasing a drug load upon exposure to ultrasound (US). Inclusion of dioleoylphosphatidylethanolamine (DOPE) in liposome membranes was previously shown to induce sonosensitivity. Long blood circulation time of the liposomal drug is required for high tumour uptake and efficient US-mediated drug delivery. In this study, blood pharmacokinetics of DOPE-based liposomal doxorubicin (DXR) were evaluated in non-tumoured mice. A markedly faster blood clearance of DXR was observed for DOPE-rich liposomes compared to Caelyx® (standard liposomal DXR). Subsequently, liposome membrane composition was altered to improve drug retention in the bloodstream, whilst maintaining sonosensitivity. Formulations with reduced blood clearance of DXR were obtained by reducing the content of DOPE from 62 to 32 or 25 mol%. These formulations showed long blood circulation time, as approximately 20% of the administered DXR dose was present in the bloodstream 24 h after intravenous injection. The reduction in liposomal DOPE content did not significantly reduce US-mediated DXR release in vitro, indicating that DOPE is a potent modulator of sonosensitivity. The novel liposome formulations, containing moderate amounts of DOPE, displayed similar blood pharmacokinetic profiles as standard liposomal DXR, but a markedly improved sonosensitivity.

  6. Complex changes in the apoptotic and cell differentiation programs during initiation of the hair follicle response to chemotherapy.

    PubMed

    Sharova, Tatyana Y; Poterlowicz, Krzysztof; Botchkareva, Natalia V; Kondratiev, Nikita A; Aziz, Ahmar; Spiegel, Jeffrey H; Botchkarev, Vladimir A; Sharov, Andrey A

    2014-12-01

    Chemotherapy has severe side effects in normal rapidly proliferating organs, such as hair follicles, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin (DXR), and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in DXR-treated hair follicles versus controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) receptors 1/2), as well as of a large number of keratin-associated protein genes, were seen after DXR treatment. Hair follicle apoptosis induced by DXR was significantly inhibited by either TRAIL-neutralizing antibody or caspase-8 inhibitor, thus suggesting a previously unreported role for TRAIL receptor signaling in mediating DXR-induced hair loss. These data demonstrate that the early phase of the hair follicle response to DXR includes upregulation of apoptosis-associated markers, as well as substantial reorganization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies toward the design of effective approaches for the management of chemotherapy-induced hair loss.

  7. Beneficial effects of carbon monoxide-releasing molecule-2 (CORM-2) on acute doxorubicin cardiotoxicity in mice: Role of oxidative stress and apoptosis

    SciTech Connect

    Soni, Hitesh; Pandya, Gaurav; Patel, Praful; Acharya, Aviseka; Jain, Mukul; Mehta, Anita A.

    2011-05-15

    Doxorubicin (DXR) has been used in variety of human malignancies for decades. Despite its efficacy in cancer, clinical usage is limited because of its cardiotoxicity, which has been associated with oxidative stress and apoptosis. Carbon monoxide-releasing molecules (CORMs) have been shown to reduce the oxidative damage and apoptosis. The present study investigated the effects of CORM-2, a fast CO-releaser, against DXR-induced cardiotoxicity in mice using biochemical, histopathological and gene expression approaches. CORM-2 (3, 10 and 30 mg/kg/day) was administered intraperitoneally (i.p.) for 10 days and terminated the study on day 11. DXR (20 mg/kg, i.p.) was injected before 72 h of termination. Mice treated with DXR showed cardiotoxicity as evidenced by elevation of serum creatine kinase (CK) and lactate dehydrogenase (LDH), tissue malondialdehyde (MDA), caspase-3 and decrease the level of total antioxidant status (TAS) in heart tissues. Pre- and post-treatment with CORM-2 (30 mg/kg, i.p.) elicited significant improvement in CK, LDH, MDA, caspase-3 and TAS levels. Histopathological studies showed that cardiac damage with DXR has been reversed with CORM-2 + DXR treatment. There was dramatic decrease in hematological count in DXR-treated mice, which has been improved with CORM-2. Furthermore, there was also elevation of mRNA expression of heme oxygenase-1, hypoxia inducible factor-1 alpha, vascular endothelial growth factor and decrease in inducible-nitric oxide synthase expression upon treatment with CORM-2 that might be linked to cardioprotection. These data suggest that CORM-2 treatment provides cardioprotection against acute doxorubicin-induced cardiotoxicity in mice and this effect may be attributed to CORM-2-mediated antioxidant and anti-apoptotic properties.

  8. Early changes in bone mineral density measured by digital X-ray radiogrammetry predict up to 20 years radiological outcome in rheumatoid arthritis

    PubMed Central

    2011-01-01

    Introduction Changes in bone mineral density (BMD) in the hand as evaluated by digital X-ray radiogrammetry (DXR) of the second to fourth metacarpal bones has been suggested to predict future joint damage in patients with rheumatoid arthritis (RA). This study's objective was to investigate whether DXR-BMD loss early in the course of the disease predicts the development of joint damage in RA patients followed for up to 20 years. Methods A total of 183 patients (115 women and 68 men) with early RA (mean disease duration, 11 months) included from 1985 to 1989 were followed prospectively (the Lund early RA cohort). Clinical and functional measures were assessed yearly. Joint damage was evaluated according to the Larsen score on radiographs of the hands and feet obtained in years 0 to 5 and years 10, 15 and 20. These radiographs were digitized, and BMD of the second to fourth metacarpal bones was evaluated by DXR. Early DXR-BMD change rate (that is, bone loss) per year calculated from the first two radiographs obtained on average 9 months apart (SD ± 4.8) were available for 135 patients. Mean values of the right and left hand were used. Results Mean early DXR-BMD loss during the first year calculated was -0.023 g/cm2 (SD ± 0.025). Patients with marked bone loss, that is, early DXR-BMD loss above the median for the group, had significantly worse progression of joint damage at all examinations during the 20-year period. Conclusions Early DXR-BMD progression rate predicted the development of joint damage evaluated according to Larsen score at year 1 and for up to 20 years in this cohort of early RA patients. PMID:21345204

  9. Chemotherapy-induced late transgenerational effects in mice.

    PubMed

    Kujjo, Loro L; Chang, Eun A; Pereira, Ricardo J G; Dhar, Shilpa; Marrero-Rosado, Brenda; Sengupta, Satyaki; Wang, Hongbing; Cibelli, Jose B; Perez, Gloria I

    2011-03-17

    To our knowledge, there is no report on long-term reproductive and developmental side effects in the offspring of mothers treated with a widely used chemotherapeutic drug such as doxorubicin (DXR), and neither is there information on transmission of any detrimental effects to several filial generations. Therefore, the purpose of the present paper was to examine the long-term effects of a single intraperitoneal injection of DXR on the reproductive and behavioral performance of adult female mice and their progeny. C57BL/6 female mice (generation zero; G0) were treated with either a single intraperitoneal injection of DXR (G0-DXR) or saline (G0-CON). Data were collected on multiple reproductive parameters and behavioral analysis for anxiety, despair and depression. In addition, the reproductive capacity and health of the subsequent six generations were evaluated. G0-DXR females developed despair-like behaviors; delivery complications; decreased primordial follicle pool; and early lost of reproductive capacity. Surprisingly, the DXR-induced effects in oocytes were transmitted transgenerationally; the most striking effects being observed in G4 and G6, constituting: increased rates of neonatal death; physical malformations; chromosomal abnormalities (particularly deletions on chromosome 10); and death of mothers due to delivery complications. None of these effects were seen in control females of the same generations. Long-term effects of DXR in female mice and their offspring can be attributed to genetic alterations or cell-killing events in oocytes or, presumably, to toxicosis in non-ovarian tissues. Results from the rodent model emphasize the need for retrospective and long-term prospective studies of survivors of cancer treatment and their offspring.

  10. L-type cardiac calcium channels in doxorubicin cardiomyopathy in rats morphological, biochemical, and functional correlations.

    PubMed Central

    Keung, E C; Toll, L; Ellis, M; Jensen, R A

    1991-01-01

    Doxorubicin (DXR) is an effective antitumor agent in a wide spectrum of neoplasms. Chronic treatment is associated with cardiomyopathy and characteristic myocardial ultrastructural changes, which include swelling of the t tubules. Accordingly, we investigated excitation-contraction coupling in cardiomyopathic rat heart resulting from chronic DXR treatment. Using the whole-cell patch clamp technique, we studied the L-type calcium channel in single cells enzymatically isolated from normal (CTRL) and DXR rat hearts. Despite similar cell dimensions, the total membrane capacitance was significantly smaller in the DXR cells (138 +/- 9 pF) than in the CTRL cells (169 +/- 11 pF) (mean +/- SEM, n = 9, P less than 0.05). The mean current and the current density-voltage relationships of the CTRL and the DXR cells were significantly different (n = 9, P less than 0.001) with the maximal peak L-type calcium current (ICa) density increased from 6.4 +/- 0.9 in CTRL cells to 10.5 +/- 2.4 microA/cm2 in the DXR cells (P less than 0.05). There was no shift either in the current-voltage relationship or the steady-state inactivation curve in the two cell groups. However, the fast time constant of inactivation was increased at a membrane voltage of -10 to 10 mV. Calcium channel antagonist equilibrium binding assays using [3H]-PN200-110 revealed no difference in the maximal receptor binding capacity (CTRL, 194 +/- 27 and DXR 211 +/- 24 fmol/mg protein; P greater than 0.05, n = 6) and in receptor affinity (CTRL, 0.15 +/- 0.05 and DXR 0.13 +/- 0.03 nM; P less than 0.05). These data suggest that a decrease in effective capacitance might be associated with t-tubular damage. Despite this decrease, ICa was increased in the DXR cells. Such an increase may result from an alteration in the properties of the calcium channels and/or recruitment of "hibernating" channels in the remaining surface and t-tubular membranes. PMID:1645752

  11. Modulating effect of synthetic statins against damage induced by doxorubicin in somatic cells of Drosophila melanogaster.

    PubMed

    Orsolin, P C; Silva-Oliveira, R G; Nepomuceno, J C

    2015-07-01

    The competitive inhibitors of HMG-CoA reductase, popularly known as statins, exert pleiotropic effects, which result from the ability of statins to inhibit the synthesis of isoprenoids, which are fundamental for the functioning of proteins responsible for intracellular signaling. Some recent studies suggest an important role associated with the use of antineoplastic atorvastatin and rosuvastatin, the statins most widely used today. In this study, the Drosophila wing spot test was used to evaluate possible protective effects of atorvastatin and rosuvastatin against damage induced by DXR. Larvae were chronically treated with negative control (ethanol 5%), positive control (DXR 0.125 mg/mL) and five different concentrations of atorvastatin and rosuvastatin. The results demonstrated absence of a mutagenic effect for the two statins tested. The analysis of the descendants co-treated with DXR and atorvastatin/rosuvastatin revealed a modulatory effect of these statins on damage induced by DXR. This effect was verified in all concentrations tested in the descendants of the ST and HB crosses treated with rosuvastatin, and only in descendants of the HB cross treated with atorvastatin. Induction of apoptosis and antioxidant activity appear to be the main mechanisms involved in reducing the frequency of mutant spots and consequent modulation of the damage induced by DXR. PMID:25846503

  12. Modulating effect of losartan potassium on the mutagenicity and recombinogenicity of doxorubicin in somatic cells of Drosophila melanogaster.

    PubMed

    Silva-Oliveira, R G; Orsolin, P C; Nepomuceno, J C

    2016-09-01

    Losartan potassium is an antihypertensive drug in the angiotensin II receptor antagonist (ARA) class. Some studies claim that, in addition to regulating blood pressure, this class of drug has anticancer properties. The objective of this study was to evaluate the genotoxic and antigenotoxic potential of losartan potassium using the SMART (Somatic Mutation and Recombination Test) assay on the somatic cells of Drosophila melanogaster, as well as the possible modulating effects of this drug, when associated with doxorubicin (DXR). Third instar larvae, descendents of standard and high bioactivation (ST and HB) crosses, were chronically treated with different concentrations of losartan potassium (0.25; 0.5; 1; 2; and 4 mM) alone or in association (co-treatment) with doxorubicin (DXR 0.125 mg/mL). The results showed an absence of a mutagenic effect of losartan potassium. In the co-treatment of losartan with DXR, the results showed that losartan is capable of reducing the number of mutant spots induced by DXR without altering the recombinogenic effect of the chemotherapeutic agent. Antiproliferative action appears to be the main mechanism involved in reducing the frequency of mutant spots and consequent modulation of alterations induced by DXR, although this parameter has not been directly assessed in this study. PMID:27394655

  13. Effect of organic tomato (Lycopersicon esculentum) extract on the genotoxicity of doxorubicin in the Drosophila wing spot test

    PubMed Central

    2009-01-01

    The consumption of organic tomatoes (ORTs) reduces the risk of harmful effects to humans and the environment caused by exposure to toxic agrochemicals. In this study, we used the somatic mutation and recombination test (SMART) of wing spots in Drosophila melanogaster to evaluate the genotoxicity of ORT and the effect of cotreatment with ORT on the genotoxicity of Doxorubicin® (DXR, a cancer chemotherapeutic agent) that is mediated by free radical formation. Standard (ST) cross larvae were treated chronically with solutions containing 25%, 50% or 100% of an aqueous extract of ORT, in the absence and presence of DXR (0.125 mg/mL), and the number of mutant spots on the wings of emergent flies was counted. ORT alone was not genotoxic but enhanced the toxicity of DXR when administered concomitantly with DXR. The ORT-enhanced frequency of spots induced by DXR may have resulted from the interaction of ORT with the enzymatic systems that catalyze the metabolic detoxification of this drug. PMID:21637658

  14. Host cells and methods for production of isobutanol

    DOEpatents

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  15. DAFNE-Light INFN-LNF Synchrotron Radiation Facility

    SciTech Connect

    Balerna, A.; Cestelli-Guidi, M.; Cimino, R.; Commisso, M.; Grilli, A.; Pietropaoli, M.; Raco, A.; Sciarra, V.; Tullio, V.; Viviani, G.; De Sio, A.; Gambicorti, L.; Hampai, D.; Pace, E.

    2010-06-23

    DAFNE-Light is the Synchrotron Radiation Facility at the INFN-Frascati National Laboratory (Rome, Italy). Three beamlines are operational, using in parasitic and dedicated mode the intense photon emission of DAFNE, a 0.51 GeV storage ring with a routinely circulating electron current higher than 1 Ampere. Two of these beamlines--the soft x-ray (DXR1) and UV (DXR2)--use one of the DAFNE wiggler magnets as synchrotron radiation source, while the third beamline SINBAD (Synchrotron Infrared Beamline At DAFNE) collects the radiation from a bending magnet. New XUV bending magnet beamlines are nowadays under construction and the low energy one (35-200 eV) will be ready for commissioning by the end of 2009. A presentation of the facility will be given together with some recent scientific results achieved at SINBAD and DXR1 beamlines.

  16. A comparative study on volatile metabolites profile of Dracaena cochinchinensis (Lour.) S.C. Chen xylem with and without resin using GC-MS.

    PubMed

    Teng, Zhongqiu; Zhang, Mei; Meng, Shiying; Dai, Rongji; Meng, Weiwei; Deng, Yulin; Huang, Luqi

    2015-11-01

    Dragon's blood is a famous traditional Chinese medicine produced from source plants under bio- or abio-stress. Dracaena cochinchinensis (Lour.) S.C. Chen xylem (DX) is one of the most important sources of the medicine. In this work, a GC-MS method was developed for analysis of the n-hexane extracts of DX with resin (DXR) and without resin (DXW). The repeatability of the method was also investigated for a metabolite comparative study of the different xylems. About 80 components were detected, 26 of which were identified in both DXR and DXN. Three sesquiterpenes (τ-cadinol, τ-muurolon and α-cadinol) were first discovered in Dracaena cochinchinensis (Lour.) S.C. Chen. The chromatographs of the two plant materials were compared and differences of compounds were found. It showed that phytosterols showed a dramatic rise in content, and sesquiterpenes were found to be synthesized in DXR. PMID:25973719

  17. A comparative study on volatile metabolites profile of Dracaena cochinchinensis (Lour.) S.C. Chen xylem with and without resin using GC-MS.

    PubMed

    Teng, Zhongqiu; Zhang, Mei; Meng, Shiying; Dai, Rongji; Meng, Weiwei; Deng, Yulin; Huang, Luqi

    2015-11-01

    Dragon's blood is a famous traditional Chinese medicine produced from source plants under bio- or abio-stress. Dracaena cochinchinensis (Lour.) S.C. Chen xylem (DX) is one of the most important sources of the medicine. In this work, a GC-MS method was developed for analysis of the n-hexane extracts of DX with resin (DXR) and without resin (DXW). The repeatability of the method was also investigated for a metabolite comparative study of the different xylems. About 80 components were detected, 26 of which were identified in both DXR and DXN. Three sesquiterpenes (τ-cadinol, τ-muurolon and α-cadinol) were first discovered in Dracaena cochinchinensis (Lour.) S.C. Chen. The chromatographs of the two plant materials were compared and differences of compounds were found. It showed that phytosterols showed a dramatic rise in content, and sesquiterpenes were found to be synthesized in DXR.

  18. Evaluation of the mutagenicity and antimutagenicity of Ziziphus joazeiro Mart. bark in the micronucleus assay

    PubMed Central

    Boriollo, Marcelo Fabiano Gomes; Resende, Marielly Reis; da Silva, Thaísla Andrielle; Públio, Juliana Yoshida; Souza, Luiz Silva; Dias, Carlos Tadeu dos Santos; de Mello Silva Oliveira, Nelma; Fiorini, João Evangelista

    2014-01-01

    The aim of this study was to evaluate the mutagenicity (clastogenicity/aneugenicity) of a glycolic extract of Ziziphus joazeiro bark (GEZJ) by the micronucleus assay in mice bone marrow. Antimutagenic activity was also assessed using treatments associated with GEZJ and doxorubicin (DXR). Mice were evaluated 24–48 h after exposure to positive (N-nitroso-N-ethylurea, NEU - 50 mg.kg−1 and DXR - 5 mg.kg−1) and negative (150 mM NaCl) controls, as well as treatment with GEZJ (0.5–2 g.kg−1), GEZJ (2 g.kg−1) + NEU and GEZJ (2 g.kg−1) + DXR. There were no significant differences in the frequencies of micronucleated polychromatic erythrocytes in mice treated with GEJZ and GEJZ + DXR compared to the negative controls, indicating that GEZJ was not mutagenic. Analysis of the polychromatic:normochromatic erythrocyte ratio revealed significant differences in the responses to doses of 0.5 g.kg−1 and 1–2 g.kg−1 and the positive control (NEU). These results indicated no systemic toxicity and moderate toxicity at lower and higher doses of GEZJ. The lack of mutagenicity and systemic toxicity in the antimutagenic assays, especially for treatment with GEZJ + DXR, suggested that phytochemical compounds in Z. joazeiro bark attenuated DXR-induced mutagenicity and the moderate systemic toxicity of a high dose of Z. joazeiro bark (2 g.kg−1). Further studies on the genotoxicity of Z. joazeiro extracts are necessary to establish the possible health risk in humans and to determine the potential as a chemopreventive agent for therapeutic use. PMID:25071409

  19. Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage

    PubMed Central

    Seiler, Kristen M; Schenhals, Erica L; von Furstenberg, Richard J; Allena, Bhavya K; Smith, Brian J; Scaria, Denny; Bresler, Michele N; Dekaney, Christopher M; Henning, Susan J

    2015-01-01

    The goals of this study were to document the proliferative response of intestinal stem cells (ISCs) during regeneration after damage from doxorubicin (DXR) and to characterize the signals responsible for ISC activation. To this end, jejuni from DXR-treated mice were harvested for histology, assessment of ISC numbers and proliferation by flow cytometry, crypt culture, and RNA analyses. Histology showed that crypt depth and width were increased 4 days after DXR. At this time point, flow cytometry on tissue collected 1 hour after EdU administration revealed increased numbers of CD24loUEA− ISCs and increased percentage of ISCs cycling. In culture, crypts harvested from DXR-treated mice were equally proliferative as those of control mice. Addition of subepithelial intestinal tissue (SET) collected 4 days after DXR elicited increased budding (1.4 ± 0.3 vs. 5.1 ± 1.0 buds per enteroid). Microarray analysis of SET collected 4 days after DXR revealed 1,030 differentially expressed transcripts. Cross comparison of Gene Ontology terms considered relevant to ISC activation pointed to 10 candidate genes. Of these the epidermal growth factor (EGF) family member amphiregulin and the BMP antagonist chordin-like 2 were chosen for further study. In crypt culture, amphiregulin alone did not elicit significant budding, but amphiregulin in combination with BMP antagonism showed marked synergism (yielding 6.3 ± 0.5 buds per enteroid). These data suggest a critical role for underlying tissue in regulating ISC behavior after damage, and point to synergism between amphiregulin and chordin-like 2 as factors which may account for activation of ISCs in the regenerative phase. PMID:25693894

  20. Digital X-ray radiogrammetry in the study of osteoporotic fractures: Comparison to dual energy X-ray absorptiometry and FRAX.

    PubMed

    Kälvesten, Johan; Lui, Li-Yung; Brismar, Torkel; Cummings, Steven

    2016-05-01

    Osteoporosis is often underdiagnosed and undertreated. Screening of post-menopausal women for clinical risk factors and/or low bone mineral density (BMD) has been proposed to overcome this. Digital X-ray radiogrammetry (DXR) estimates hand BMD from standard hand X-ray images and have shown to predict fractures and osteoporosis. Recently, digital radiology and the internet have opened up the possibility of conducting automated opportunistic screening with DXR in post-fracture care or in combination with mammography. This study compared the performance of DXR with FRAX® and DXA in discriminating major osteoporotic fracture (MOF) (hip, clinical spine, forearm or shoulder), hip fracture and femoral neck osteoporosis. This prospective cohort study was conducted on 5278 women 65years and older in the Study of Osteoporotic Fractures (SOF) cohort. Baseline hand X-ray images were analyzed and fractures were ascertained during 10years of follow up. Age-adjusted area under receiver operating characteristic curve (AUC) for MOF and hip fracture and for femoral neck osteoporosis (DXA FN BMD T-score ≤-2.5) was used to compare the methods. Sensitivity to femoral neck osteoporosis at equal selection rates was tabulated for FRAX and DXR. DXR-BMD, FRAX (no BMD) and lumbar spine DXA BMD were all similar in fracture discriminative performance with an AUC around 0.65 for MOF and 0.70 for hip fractures for all three methods. As expected femoral neck DXA provided fracture discrimination superior both to other BMD measurements and to FRAX. AUC for selection of patients with femoral neck osteoporosis was higher with DXR-BMD, 0.76 (0.74-0.77), than with FRAX, 0.69 (0.67-0.71), (p<0.0001). In conclusion, DXR-BMD discriminates incident fractures to a similar degree as FRAX and predicts femoral neck osteoporosis to a larger degree than FRAX. DXR shows promise as a method to automatically flag individuals who might benefit from an osteoporosis assessment. PMID:26921822

  1. Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma

    PubMed Central

    Villar, Victor Hugo; Vögler, Oliver; Barceló, Francisca; Martín-Broto, Javier; Martínez-Serra, Jordi; Ruiz-Gutiérrez, Valentina; Alemany, Regina

    2016-01-01

    Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5–50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6–9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10–15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS. PMID:27219337

  2. Identification of sialylated glycoproteins in Doxorubicin-treated hepatoma cells with glycoproteomic analyses.

    PubMed

    Azuma, Kanako; Serada, Satoshi; Takamatsu, Shinji; Terao, Naoko; Takeishi, Shunsaku; Kamada, Yoshihiro; Naka, Tetsuji; Miyoshi, Eiji

    2014-11-01

    Sialylation is one of the most important types of glycosylation involved in carcinogenesis and establishment of cancer stemness. We previously showed that increased sialylation is a characteristic glycan change in cancer stem cells (CSCs) from hepatocellular carcinoma. However, the identities of glycoproteins targeted for sialylation remain unknown. In the present study, we identified glycoproteins targeted for sialylation in doxorubicin (DXR)-treated hepatocarcinoma cell line, Huh7, using glycoproteomic analyses. Since CSCs constitute a small subset of cells within carcinoma cell lines, it is difficult to identify sialylated proteins using general glycoproteomic strategies. It is known that treatment with anticancer drug can condense CSCs, we used DXR to concentrate CSCs. In DXR-treated Huh7 cells, isobaric tag for relative and absolute quantitation (iTRAQ) analysis identified 17 sialylated glycoproteins. Most of the identified glycoproteins were cancer-associated proteins. Furthermore, two proteins of approximately 70 kDa were detected using Sambucus sieboldoana agglutinin (SSA) blot analysis and identified as beta-galactosidase and alpha-2-HS-glycoprotein (fetuin-A) by SSA precipitation followed by liquid chromatography-tandem mass spectrometry analyses. Sialylation levels of fetuin-A were increased in DXR-treated Huh7 cell lysates. These changes in sialylation of glycoproteins might be involved in the establishment of cancer stemness.

  3. Tumor-Priming Smoothened Inhibitor Enhances Deposition and Efficacy of Cytotoxic Nanoparticles in a Pancreatic Cancer Model.

    PubMed

    Roy Chaudhuri, Tista; Straubinger, Ninfa L; Pitoniak, Rosemarie F; Hylander, Bonnie L; Repasky, Elizabeth A; Ma, Wen Wee; Straubinger, Robert M

    2016-01-01

    Most pancreatic adenocarcinoma patients present with unresectable disease and benefit little from chemotherapy. Poor tumor perfusion and vascular permeability limit drug deposition. Previous work showed that Smoothened inhibitors of hedgehog signaling (sHHI) promote neovascularization in spontaneous mouse models of pancreatic cancer (PaCA) and enhance tumor permeability to low-molecular weight compounds. Here, we tested the hypothesis that sHHI can enhance tumor deposition and efficacy of drug-containing nanoparticles consisting of 80 to 100 nm sterically-stabilized liposomes (SSL) containing doxorubicin (SSL-DXR). SCID mice bearing low-passage patient-derived PaCA xenografts (PDX) were pretreated p.o. for 10 days with 40 mg/kg/d NVP-LDE225 (erismodegib), followed by i.v. SSL-DXR. Microvessel density, permeability, perfusion, and morphology were compared with untreated controls, as was SSL deposition and therapeutic efficacy. The sHHI alone affected tumor growth minimally, but markedly increased extravasation of nanoparticles into adenocarcinoma cell-enriched regions of the tumor. Immunostaining showed that sHHI treatment decreased pericyte coverage (α-SMA(+)) of CD31(+) vascular endothelium structures, and increased the abundance of endothelium-poor (CD31(-)) basement membrane structures (collagen IV(+)), suggesting increased immature microvessels. SSL-DXR (15 mg/kg) administered after sHHI pretreatment arrested tumor volume progression and decreased tumor perfusion/permeability, suggesting an initial vascular pruning response. Compared with controls, one cycle of 10-day sHHI pretreatment followed by 6 mg/kg SSL-DXR doubled median tumor progression time. Three cycles of treatment with sHHI and SSL-DXR, with a 10-day between-cycle drug holiday, nearly tripled median tumor progression time. Based upon these data, short-term sHHI treatment sequenced with nanoparticulate drug carriers constitutes a potential strategy to enhance efficacy of pancreatic cancer therapy

  4. Tumor-Priming Smoothened Inhibitor Enhances Deposition and Efficacy of Cytotoxic Nanoparticles in a Pancreatic Cancer Model.

    PubMed

    Roy Chaudhuri, Tista; Straubinger, Ninfa L; Pitoniak, Rosemarie F; Hylander, Bonnie L; Repasky, Elizabeth A; Ma, Wen Wee; Straubinger, Robert M

    2016-01-01

    Most pancreatic adenocarcinoma patients present with unresectable disease and benefit little from chemotherapy. Poor tumor perfusion and vascular permeability limit drug deposition. Previous work showed that Smoothened inhibitors of hedgehog signaling (sHHI) promote neovascularization in spontaneous mouse models of pancreatic cancer (PaCA) and enhance tumor permeability to low-molecular weight compounds. Here, we tested the hypothesis that sHHI can enhance tumor deposition and efficacy of drug-containing nanoparticles consisting of 80 to 100 nm sterically-stabilized liposomes (SSL) containing doxorubicin (SSL-DXR). SCID mice bearing low-passage patient-derived PaCA xenografts (PDX) were pretreated p.o. for 10 days with 40 mg/kg/d NVP-LDE225 (erismodegib), followed by i.v. SSL-DXR. Microvessel density, permeability, perfusion, and morphology were compared with untreated controls, as was SSL deposition and therapeutic efficacy. The sHHI alone affected tumor growth minimally, but markedly increased extravasation of nanoparticles into adenocarcinoma cell-enriched regions of the tumor. Immunostaining showed that sHHI treatment decreased pericyte coverage (α-SMA(+)) of CD31(+) vascular endothelium structures, and increased the abundance of endothelium-poor (CD31(-)) basement membrane structures (collagen IV(+)), suggesting increased immature microvessels. SSL-DXR (15 mg/kg) administered after sHHI pretreatment arrested tumor volume progression and decreased tumor perfusion/permeability, suggesting an initial vascular pruning response. Compared with controls, one cycle of 10-day sHHI pretreatment followed by 6 mg/kg SSL-DXR doubled median tumor progression time. Three cycles of treatment with sHHI and SSL-DXR, with a 10-day between-cycle drug holiday, nearly tripled median tumor progression time. Based upon these data, short-term sHHI treatment sequenced with nanoparticulate drug carriers constitutes a potential strategy to enhance efficacy of pancreatic cancer therapy.

  5. Sodium glucose cotransporter 1 ligand BLF501 as a novel tool for management of gastrointestinal mucositis

    PubMed Central

    2014-01-01

    Background Recent studies demonstrated that engagement of sodium glucose transporter 1 (SGLT-1) by orally administered D-glucose protects the intestinal mucosa from lipopolysaccharide (LPS)-induced injury. We tested whether SGLT-1 engagement might protect the intestinal mucosa from doxorubicin (DXR)- and 5-fluorouracil (5-FU)-induced injury in animal models mimicking acute or chronic mucositis. Methods Mice were treated intraperitoneally with DXR, alone or in combination with 5-FU, and orally with BLF501, a glucose-derived synthetic compound with high affinity for SGLT-1. Intestinal mucosal epithelium integrity was assessed by histological analysis, cellular proliferation assays, real-time PCR gene expression assays and Western blot assays. Student’s t-test (paired two-tailed) and χ2 analyses were used for comparisons between groups. Differences were considered significant at p < 0.05. Results BLF501 administration in mice treated with DXR and/or 5-FU decreased the injuries to the mucosa in terms of epithelial integrity and cellular proliferative ability. Co-treatment with BLF501 led to a normal expression and distribution of both zonula occludens-1 (ZO-1) and beta-catenin, which were underexpressed after treatment with either chemotherapeutic agent alone. BLF501 administration also restored normal expression of caspase-3 and ezrin/radixin/moesin (ERM), which were overexpressed after treatment with DXR and 5-FU. In SGLT1-/- mice, BLF501 had no detectable effects. BLF501 administration in wild-type mice with growing A431 tumors did not modify antitumor activity of DXR. Conclusions BLF501-induced protection of the intestinal mucosa is a promising novel therapeutic approach to reducing the severity of chemotherapy-induced mucositis. PMID:24495286

  6. Effects of vitamin D on kidney histology and trpv1 channels in doxorubicin-induced nephropathy.

    PubMed

    Gurel, Ali; Atli, Hasan; Kaya, Nalan; Onalan, Ebru; Kuloglu, Tuncay; Aygen, Bilge

    2015-01-01

    Doxorubicin (DXR) is an antineoplastic agent of the anthracycline group, and may show nephrotoxic effects in animal models and humans. We investigated changes in kidney tissue following doxorubicin treatment and the effects of vitamin D on kidney tissue and TRPV1 channels. In this study, 24 adult male Wistar Albino rats were used. The animals were divided into four groups of six animals. During the 14-day experiment period, Group I did not have any application. 200 IU/day cholecalciferol was administered orally to Group II. Group III received 10 mg/kg single dose of DXR intraperitoneally (IP); and Group IV had a single 10 mg/kg dose of IP DXR and 200 IU/day of oral cholecalciferol. At the end of the experiment, the rats were decapitated, and their kidney tissues were removed. TRPV1 expression and apoptosis were detected in the tissue section by using immunohistochemical, TUNEL and real time-PCR (RT-PCR) techniques. The findings were examined and photographed with BH2 Olympus photomicroscope. As result of immunohistochemical staining, RT-PCR and examination with light microscope, it was found that the TRPV 1 immunoreactivity of the DXR group decreased in comparison with the control group, and the vitamin D application did not reverse this effect. Apoptosis detected by the TUNEL method tended to increase in the doxorubicin group and was relatively reversed with the administration of vitamin D. Tissue malondialdehyde (MDA) levels were observed to correlate with the findings of apoptosis. This study showed that vitamin D has anti- apoptotic and antioxidant effects on kidney tissue after DXR-induced injury.

  7. Effects of vitamin D on kidney histology and trpv1 channels in doxorubicin-induced nephropathy

    PubMed Central

    Gurel, Ali; Atli, Hasan; Kaya, Nalan; Onalan, Ebru; Kuloglu, Tuncay; Aygen, Bilge

    2015-01-01

    Doxorubicin (DXR) is an antineoplastic agent of the anthracycline group, and may show nephrotoxic effects in animal models and humans. We investigated changes in kidney tissue following doxorubicin treatment and the effects of vitamin D on kidney tissue and TRPV1 channels. In this study, 24 adult male Wistar Albino rats were used. The animals were divided into four groups of six animals. During the 14-day experiment period, Group I did not have any application. 200 IU/day cholecalciferol was administered orally to Group II. Group III received 10 mg/kg single dose of DXR intraperitoneally (IP); and Group IV had a single 10 mg/kg dose of IP DXR and 200 IU/day of oral cholecalciferol. At the end of the experiment, the rats were decapitated, and their kidney tissues were removed. TRPV1 expression and apoptosis were detected in the tissue section by using immunohistochemical, TUNEL and real time-PCR (RT-PCR) techniques. The findings were examined and photographed with BH2 Olympus photomicroscope. As result of immunohistochemical staining, RT-PCR and examination with light microscope, it was found that the TRPV 1 immunoreactivity of the DXR group decreased in comparison with the control group, and the vitamin D application did not reverse this effect. Apoptosis detected by the TUNEL method tended to increase in the doxorubicin group and was relatively reversed with the administration of vitamin D. Tissue malondialdehyde (MDA) levels were observed to correlate with the findings of apoptosis. This study showed that vitamin D has anti- apoptotic and antioxidant effects on kidney tissue after DXR-induced injury. PMID:26550293

  8. Multimodal transfer of MDR by exosomes in human osteosarcoma.

    PubMed

    Torreggiani, Elena; Roncuzzi, Laura; Perut, Francesca; Zini, Nicoletta; Baldini, Nicola

    2016-07-01

    Exosomes are extracellular vesicles released by both normal and tumour cells which are involved in a new intercellular communication pathway by delivering cargo (e.g., proteins, microRNAs, mRNAs) to recipient cells. Tumour-derived exosomes have been shown to play critical roles in different stages of tumour growth and progression. In this study, we investigated the potential role of exosomes to transfer the multidrug resistance (MDR) phenotype in human osteosarcoma cells. Exosomes were isolated by differential centrifugation of culture media from multidrug resistant human osteosarcoma MG-63DXR30 (Exo/DXR) and MG-63 parental cells (Exo/S). Exosome purity was examined by transmission electron microscopy and confirmed by immunoblot analysis for the expression of specific exosomal markers. Our data showed that exosomes derived from doxorubicin-resistant osteosarcoma cells could be taken up into secondary cells and induce a doxorubicin-resistant phenotype. The incubation of osteosarcoma cells with Exo/DXR decreased the sensitivity of parental cells to doxorubicin, while exposure with Exo/S was ineffective. In addition, we demonstrated that Exo/DXR expressed higher levels of MDR-1 mRNA and P-glycoprotein compared to Exo/S (p=0.03). Interestingly, both MDR-1 mRNA and P-gp increased in MG-63 cells after incubation with Exo/DXR, suggesting this as the main mechanism of exosome-mediated transfer of drug resistance. Our findings suggest that multidrug resistant osteosarcoma cells are able to spread their ability to resist the effects of doxorubicin treatment on sensitive cells by transferring exosomes carrying MDR-1 mRNA and its product P-glycoprotein. PMID:27176642

  9. Pharmaceutical Approval Update.

    PubMed

    Kaufman, Michele B

    2016-08-01

    Venetoclax (Venclexta) for chronic lymphocytic leukemia; riboflavin 5'-phosphate solutions (Photrexa Viscous and Photrexa) for progressive keratoconus; and pimavanserin (Nuplazid) for Parkinson's disease psychosis. PMID:27504063

  10. Biosynthesis of riboflavin. Characterization of the product of the deaminase.

    PubMed

    Nielsen, P; Bacher, A

    1981-12-15

    The 2'5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate deaminase was partially purified from cell extracts of Candida guilliermondii ATCC 9058. The enzyme requires Mg2+ for activity. Maximal activity was observed at pH 7,3. The enzyme converts its substrate, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, to 2,5-diamino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate. This labile compound was treated with diacetyl and the resulting 6,7-dimethyl-8-ribityllumazine 5'-phosphate was identified by comparison with a synthetic sample. PMID:7317443

  11. The association of ribulose-1,5-bisphosphate carboxylase with phosphoriboisomerase and phosphoribulokinase.

    PubMed

    Sainis, J K; Harris, G C

    1986-09-30

    RuBPCase from peas showed Ribose-5-phosphate and Ribulose-5-phosphate dependent CO2 fixation when purified on sucrose gradients or by conventional methods. If purification was done in the presence of 20 mM MgCl2 and 20-25 mM NaHCO3 RuBPCase showed higher Ribose-5-phosphate and Ribulose-5-phosphate dependent CO2 fixation rates. Partially purified phosphoriboisomerase, phosphoribulokinase and RuBPCase from spinach could be reassociated in vitro.

  12. Ozone Ameliorates Doxorubicine-Induced Skin Necrosis - results from an animal model.

    PubMed

    Kesik, Vural; Yuksel, Ramazan; Yigit, Nuri; Saldir, Mehmet; Karabacak, Ercan; Erdem, Galip; Babacan, Oguzhan; Gulgun, Mustafa; Korkmazer, Nadir; Bayrak, Ziya

    2016-09-01

    Doxorubicin (DXR) extravasation result with serious morbidity like skin ulceration and necrosis. The purpose of this study is to determine the protective effects of ozone, olive oil, dimethyl sulfoxide (DMSO), and coenzyme Q10 in the treatment of DXR-induced skin ulcers on rats. After an intradermal injection of DXR on a basis of an animal extravasation model, the materials were topically applied. The ulcer sizes were measured, and a punch biopsy was taken from the extravasation site in which the skin ulcers formed at the end of the experiment. The samples were analyzed for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL1β), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) enzymes, and examined histopathologically. The ulcer sizes clearly decreased in the study groups, including DMSO, olive oil, ozone plus coenzyme Q10, and ozone plus olive oil groups in comparison with the control group with the exception of the coenzyme Q10 group. The malondialdehyde levels were lower in the DMSO, olive oil, ozone plus olive oil, and ozone plus coenzyme Q10 groups than they were in the control group, but they were not significantly different. The TNF-α level was lower in the DMSO, ozone plus olive oil, coenzyme Q10, and ozone plus coenzyme Q10 groups in comparison with the control group. There was no significant change in the SOD, GSH-Px, and IL1β levels in the study groups in comparison with the control and the sham groups. The ozone plus olive oil group could be considered to be an alternate therapy for skin ulcers due to DXR extravasation. PMID:26286933

  13. Genotoxic and Antigenotoxic Potential of Momordica charantia Linn (Cucurbitaceae) in the Wing Spot Test of Drosophila melanogaster.

    PubMed

    Guterres, Zaira Rosa; Zanetti, Thalita Alves; Sennes-Lopes, Tiago Felipe; da Silva, Ana Francisca Gomes

    2015-10-01

    Momordica charantia, popularly known as bitter melon, is a plant widely used in ethnobotanical medicine. It has antibacterial, antifungal, anthelmintic, antidiabetic, antiviral, and antimalarial activities, among others. The goal of this study was to evaluate the genotoxic and/or antigenotoxic activity of the aqueous extracts obtained from the aerial parts and fruit of this plant by means of the Drosophila melanogaster wing spot test. Third-stage larvae that obtained standard (ST) cross and high bioactivation (HB) cross were treated with aqueous extracts of the aerial parts (IQA) and fruit (IQF) of M. charantia, following two protocols (genotoxicity and antigenotoxicity). The aqueous extracts are not genotoxic in lower concentrations. The frequencies of mutant spots observed in the descendants of the ST and HB crosses treated with doxorubicin (DXR) alone were 8.65 and 9.25, respectively, whereas in those cotreated with IQA and DXR, the frequencies ranged from 15.90 to 29 in the ST cross and from 15.05 to 24.78 in the HB cross. In cotreatment with IQF, the frequencies ranged from 30.10 to 30.65 in the ST cross and from 13.60 to 14.50 in the HB cross, whereas the frequencies obtained with DXR were 32.50 in the ST cross and 26.00 in the HB cross. In conclusion, the IQA has a synergistic effect, enhancing the genotoxicity of DXR in the ST cross and the HB cross, whereas the IQF has antigenotoxic effects in the HB cross.

  14. Effects of leflunomide and methotrexate in rheumatoid arthritis detected by digital X-ray radiogrammetry and computer-aided joint space analysis.

    PubMed

    Pfeil, Alexander; Lippold, Julia; Eidner, Thorsten; Lehmann, Gabriele; Oelzner, Peter; Renz, Diane M; Hansch, Andreas; Wolf, Gunter; Hein, Gert; Kaiser, Werner A; Böttcher, Joachim

    2009-01-01

    The aim of the present study was to evaluate the effect of long-term leflunomide and methotrexate (MTX) therapy during the course of rheumatoid arthritis (RA) estimated by digital X-ray radiogrammetry (DXR) and computer-aided joint space analysis (CAJSA) as diagnostic tools for the quantification of disease-related periarticular osteoporosis and joint space narrowing. Fourty matchable patients with verified RA were treated with leflunomide or MTX during an observation period of 2.5 years. All patients underwent complete computerized calculations of bone mineral density (BMD) and metacarpal index (MCI) by DXR as well as semi-automated measurements of joint space widths (JSW) at the metacarpophalangeal articulations (MCP, thumb to small finger) and proximal interphalangeal joints (PIP, index finger to small finger) using digitized hand radiographs. DXR-BMD revealed an increase of 0.4% (leflunomide-group) versus a reduction of -9.1% (MTX-group). Regarding DXR-MCI, a reduction of -1.1% (leflunomide-group) and -5.3% (MTX-group) was observed. The CAJSA parameters showed a decline of -2.7% (JSW-MCP) versus -2.1% (JSW-PIP) in patients treated with leflunomide. An accentuated joint space narrowing was revealed (JSW-MCP: -5.7%; JSW-PIP: -6.2%) in the MTX group. Digital X-ray radiogrammetry and CAJSA could discriminate the influence of different therapeutic regimes on periarticular osteoporosis and joint space narrowing showing a less accentuated radiographic progression in patients treated with leflunomide.

  15. Modulating effect of simvastatin on the DNA damage induced by doxorubicin in somatic cells of Drosophila melanogaster.

    PubMed

    Orsolin, P C; Silva-Oliveira, R G; Nepomuceno, J C

    2016-04-01

    Simvastatin is an antilipemic drug that promotes inhibition of HMG-CoA reductase. Simvastatin can also inhibit the formation of other products, such as isoprenoids, conferring additional benefits to this drug, which include antiproliferative, anti-invasive and pro-apoptotic effects. This study was carried out with the aim of evaluating the mutagenic/recombinogenic effect of simvastatin as well as the possible modulatory effects of this statin on the DNA damage induced by doxorubicin (DXR). This analysis was performed using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. To study these effects, larvae descendants of both crosses (ST and HB) were chronically treated with five concentrations of simvastatin, separately and in association with DXR. The results revealed no mutagenic/recombinogenic effect of simvastatin for any of the concentrations tested. A modulating effect of simvastatin was also observed on DNA damage induced by DXR. The reduction of total mutant frequency was observed for spots from descendants of both crosses, but the inhibition was more effective in descendants from the standard cross (ST). It is believed that this modulating effect is mainly associated with the antioxidant activity of this class of drugs, although this parameter has not been directly assessed in this study. PMID:26829615

  16. Evaluation of antigenotoxic effects of plant flavonoids quercetin and rutin on HepG2 cells.

    PubMed

    Barcelos, Gustavo R M; Grotto, Denise; Angeli, José Pedro F; Serpeloni, Juliana M; Rocha, Bruno A; Bastos, Jairo K; Barbosa, Fernando

    2011-09-01

    The flavonoid quercetin and its derivative rutin were investigated for genotoxicity/antigenotoxicity activity in human hepatoma HepG2 cells using the comet assay. The extract cytotoxicity was evaluated using the trypan blue exclusion dye method with quercetin and rutin concentrations ranging from 0.1 to 200.0 μg/mL of culture medium. Three minor non-cytotoxic concentrations were chosen to evaluate the genotoxicity and antigenotoxicity of the flavonoids (0.1, 1.0 and 5.0 μg/mL) through comet assay. The cultures were treated with three different concentrations of rutin or quercetin (genotoxicity) or their association with Aflatoxin B1 (AFB1), methyl methanesulfonate (MMS) or doxorubicin (DXR) (antigenotoxicity test) in three protocols: pre-treatment, simultaneous treatment and post-treatment. The cell cultures were also treated with 1% DMSO (control group), AFB1, MMS and DXR (positive-control). Statistical analyses were performed using ANOVA and Dunnett's test (p ≤ 0.05). Quercetin at concentrations higher than 10.0 μg/mL or rutin higher than 50.0 μg/mL exhibited a cytotoxic effect on the cells, showing that quercetin is more cytotoxic than rutin. Furthermore, neither compound was able to induce genotoxicity in the concentrations evaluated. On the other hand, both flavonoids reduced DNA damage induced by AFB1, MMS and DXR in all treatment protocols.

  17. Enzymatic synthesis of 3-deoxy-d-manno-octulosonic acid (KDO) and its application for LPS assembly.

    PubMed

    Wen, Liuqing; Zheng, Yuan; Li, Tiehai; Wang, Peng George

    2016-06-15

    The studies of 3-deoxy-d-manno-octulosonic acid (KDO) have been hindered due to its limited availability. Herein, an efficient enzymatic system for the facile synthesis of KDO from easy-to-get starting materials is described. In this one-pot three-enzyme (OPME) system, d-ribulose 5-phosphate, which was prepared from d-xylose, was employed as starting materials. The reaction process involves the isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by d-arabinose 5-phosphate isomerase (KdsD), the aldol condensation of d-arabinose 5-phosphate and phosphoenolpyruvate (PEP) catalyzed by KDO 8-phosphate synthetase (KdsA), and the hydrolysis of KDO-8-phosphate catalyzed by KDO 8-phosphate phosphatase (KdsC). By using this OPME system, 72% isolated yield was obtained. The obtained KDO was further transferred to lipid A by KDO transferase from Escherichia coli (WaaA).

  18. Individual vitamin B6 contents in selected Japanese sushi toppings.

    PubMed

    Do, Huong T V; Yagi, Toshiharu

    2012-03-01

    The contents of six natural vitamin B(6) forms in popular Japanese sushi toppings were determined by a 4-pyridoxolactone-conversion HPLC method. The half-baked bonito exhibited the highest total vitamin B(6) content and the northern shrimp sashimi the lowest. Pyridoxal 5'-phosphate plus pyridoxal was predominant in nine samples, and pyridoxamine 5'-phosphate plus pyridoxamine in two other samples. Pyridoxine 5'-phosphate plus pyridoxine was minor. The raw meats (sashimi) of fatty seawater fishes contain a lot of pyridoxal 5'-phosphate and/or pyridoxamine 5'-phosphate. Five portions of sushi with 20 g of fatty seawater sashimi toppings would supply with vitamin B(6) recommended by the Japanese Recommended Daily Allowance.

  19. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles.

    PubMed

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

  20. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

    PubMed Central

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

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

    DOEpatents

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

    2001-01-01

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

  2. Engineered microorganisms capable of producing target compounds under anaerobic conditions

    DOEpatents

    Buelter, Thomas; Meinhold, Peter; Feldman, Reid M. Renny; Hawkins, Andrew C.; Urano, Jun; Bastian, Sabine; Arnold, Frances

    2012-01-17

    The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

  3. Heavy metal impurities impair the spectrophotometric assay of ribulose bisphosphate carboxylase activity.

    PubMed

    Walbot, V

    1977-01-01

    An inverse relationship between the concentration of ribose 5-phosphate and apparent ribulose bisphosphate carboxylase activity was observed. The Lilley-Walker assay spectrophotometric assay, in which the 3-phosphoglyceric acid-dependent oxidation of reduced pyridine nucleotide is measured, is shown to be highly sensitive to inhibition by heavy metals. Analysis of the purity of reagents showed that ribose 5-phosphate is often contaminated with lead in sufficient quantity to impair the assay. This noncompetitive inhibition by ribose 5-phosphate is independent of the competitive inhibition of this substrate as an ATP sink as described by Slabas and Walker. A method for checking reagent purity and removing heavy metal contaminants is described.

  4. Evaluation of the genotoxic and antigenotoxic effects after acute and subacute treatments with açai pulp (Euterpe oleracea Mart.) on mice using the erythrocytes micronucleus test and the comet assay.

    PubMed

    Ribeiro, Juliana Carvalho; Antunes, Lusânia Maria Greggi; Aissa, Alexandre Ferro; Darin, Joana D'arc Castania; De Rosso, Veridiana Vera; Mercadante, Adriana Zerlotti; Bianchi, Maria de Lourdes Pires

    2010-01-01

    Açai, the fruit of a palm native to the Amazonian basin, is widely distributed in northern South America, where it has considerable economic importance. Whereas individual polyphenolics compounds in açai have been extensively evaluated, studies of the intact fruit and its biological properties are lacking. Therefore, the present study was undertaken to investigate the in vivo genotoxicity of açai and its possible antigenotoxicity on doxorubicin (DXR)-induced DNA damage. The açai pulp doses selected were 3.33, 10.0 and 16.67g/kg b.w. administered by gavage alone or prior to DXR (16mg/kg b.w.) administered by intraperitoneal injection. Swiss albino mice were distributed in eight groups for acute treatment with açai pulp (24h) and eight groups for subacute treatment (daily for 14 consecutive days) before euthanasia. The negative control groups were treated in a similar way. The results of chemical analysis suggested the presence of carotenoids, anthocyanins, phenolic, and flavonoids in açai pulp. The endpoints analyzed were micronucleus induction in bone marrow and peripheral blood cells polychromatic erythrocytes, and DNA damage in peripheral blood, liver and kidney cells assessed using the alkaline (pH >13) comet assay. There were no statistically significant differences (p>0.05) between the negative control and the groups treated with the three doses of açai pulp alone in all endpoints analyzed, demonstrating the absence of genotoxic effects. The protective effects of açai pulp were observed in both acute and subacute treatments, when administered prior to DXR. In general, subacute treatment provided greater efficiency in protecting against DXR-induced DNA damage in liver and kidney cells. These protective effects can be explained as the result of the phytochemicals present in açai pulp. These results will be applied to the developmental of food with functional characteristics, as well as to explore the characteristics of açai as a health promoter.

  5. N-phenylglycolhydroxamate production by the action of transketolase on nitrosobenzene.

    PubMed Central

    Corbett, M D; Chipko, B R

    1977-01-01

    The incubation of nitrosobenzene with yeast transketolase and D-xylulose 5-phosphate resulted in the production of N-phenylglycolhydroxamic acid. The addition of D-ribose 5-phosphate decreased the amount of hydroxamic acid that was produced. This conversion of nitrosobenzene into the glycollic acid-derived hydroxamic acid was shown to be an enzymic process, and a chemical mechanism for the conversion was proposed. PMID:921749

  6. Modulatory effects of Tabebuia impetiginosa (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster

    PubMed Central

    2009-01-01

    The wing Somatic Mutation and Recombination Test (SMART) in D. melanogaster was used to study genotoxicity of the medicinal plant Tabebuia impetiginosa. Lapachol (naphthoquinone) and β-lapachone (quinone) are the two main chemical constituents of T. impetiginosa. These compounds have several biological properties. They induce apoptosis by generating oxygen-reactive species, thereby inhibiting topoisomerases (I and II) or inducing other enzymes dependent on NAD(P)H:quinone oxidoreductase 1, thus affecting cell cycle checkpoints. The SMART was used in the standard (ST) version, which has normal levels of cytochrome P450 (CYP) enzymes, to check the direct action of this compound, and in the high bioactivation (HB) version, which has a high constitutive level of CYP enzymes, to check for indirect action in three different T. impetiginosa concentrations (10%, 20% or 40% w/w). It was observed that T. impetiginosa alone did not modify the spontaneous frequencies of mutant spots in either cross. The negative results observed prompted us to study this phytotherapeuticum in association with the reference mutagen doxorubicin (DXR). In co-treated series, T. impetiginosa was toxic in both crosses at higher concentration, whereas in the HB cross, it induced a considerable potentiating effect (from ~24.0 to ~95.0%) on DXR genotoxity. Therefore, further research is needed to determine the possible risks associated with the exposure of living organisms to this complex mixture. PMID:21637695

  7. Enhancement of Cytotoxicity of Three Apoptosis-inducing Agents Against Human Oral Squamous Cell Carcinoma Cell Line by Benzoxazinotropone.

    PubMed

    Tomikoshi, Yukiko; Nomura, Maki; Okudaira, Noriyuki; Sakagami, Hiroshi; Wakabayashi, Hidetsugu

    Tumor-specificity (TS) and anti-inflammatory activity of benzo[b]cyclohept[e][1,4]oxazin-6(11H)-one, generally known as benzoxazinotropone (BOT), have been reported. In order to find a new biological activity, the combination effect of BOT and three apoptosis-inducing agents was investigated. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and five human oral normal cells (gingival fibroblasts, periodontal ligament fibroblasts, pulp cells, oral keratinocytes and primary gingival epithelial cells) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. TS was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal oral cells to the one against OSCC cell lines. Synergy was evaluated by CompuSyn software program. Expression of cleaved forms of poly ADP-ribose polymerase and caspsase-3 was evaluated by western blot analysis. BOT induced activation of caspase 3, suggesting the apoptosis induction in HSC-2 OSCC cells. BOT enhanced the cytotoxicity of doxorubicin (DXR) additively and that of curcumin and resveratrol synergistically. On the other hand, BOT did not enhance, but rather inhibit the cytotoxicity of DXR against normal keratinocytes. The present study suggests that BOT may enhance the anti-tumor activity of apoptosis-inducing agents, while reducing its cytotoxicity against normal cells. PMID:27566085

  8. Modulatory Effect of Betulinic Acid on the Genotoxicity Induced by Different Mutagens in V79 Cells

    PubMed Central

    Acésio, Nathália Oliveira; de Oliveira, Pollyanna Francielli; Mastrocola, Daiane Fernanda Pereira; Lima, Ildercílio Mota de Souza; Munari, Carla Carolina; Sato, Vânia Luiza Ferreira Lucatti; Souza, Andressa Aparecida Silva; Flauzino, Lúzio Gabriel Bocalon; Cunha, Wilson Roberto; Tavares, Denise Crispim

    2016-01-01

    Betulinic acid (BA) is a pentacyclic triterpene that can be isolated from many medicinal plants around the world. The aim of this study was to evaluate the genotoxic potential of BA and its effect on the genotoxicity induced by different mutagens in V79 cells using the cytokinesis-block micronucleus assay. Different BA concentrations were combined with methyl methanesulfonate (MMS), doxorubicin (DXR), camptothecin (CPT), and etoposide (VP-16). The frequencies of micronuclei in cultures treated with different BA concentrations did not differ from those of the negative control. Treatment with BA and MMS resulted in lower micronucleus frequencies than those observed for cultures treated with MMS alone. On the other hand, a significant increase in micronucleus frequencies was observed in cultures treated with BA combined with DXR or VP-16 when compared to these mutagens alone. The results showed no effect of BA on CPT-induced genotoxicity. Therefore, BA was not genotoxic under the present experimental conditions and exerted a different influence on the genotoxicity induced by different mutagens. The modulatory effect of BA depends on the type of mutagen and concentrations used. PMID:27195016

  9. The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart.

    PubMed Central

    West, S M; Price, N C

    1989-01-01

    The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred. PMID:2775204

  10. Glycation of plasma lipoprotein lipid membrane and screening for lipid glycation inhibitor.

    PubMed

    Nakagawa, Kiyotaka; Ibusuki, Daigo; Yamashita, Shinji; Miyazawa, Teruo

    2008-04-01

    We recently reported that phosphatidylethanolamine (PE)-linked Amadori product (Amadori-PE) increased abnormally in diabetic plasma. However, the glycation mechanism of human plasma low-density lipoprotein (LDL) is still unclear. Moreover, lipid glycation inhibitors have yet to be discovered. In this study, we compared the glycation kinetics of LDL lipid and LDL protein in vitro and screened lipid glycation inhibitors. LDL-PE was converted to Amadori-PE followed by LDL protein (apoB) glycation. Pyridoxal 5'-phosphate could easily react with PE before the glucose-PE reaction, and the PE-pyridoxal 5'-phosphate adduct was detected in human red blood cells. Pyridoxal 5'-phosphate can be used in diabetes prevention. PMID:18448833

  11. Strategies for improving chemotherapeutic delivery to solid tumors mediated by vascular permeability modulation

    NASA Astrophysics Data System (ADS)

    Roy Chaudhuri, Tista

    An essential mode of distribution of blood-borne chemotherapeutic agents within a solid tumor is via the micro-circulation. Poor tumor perfusion, because of a lack of functional vasculature or a lack of microvessels, as well as low tumor vascular permeability, can prevent adequate deposition of even low molecular-weight agents into the tumor. The modulation of tumor vascular function and density can provides numerous strategies for improving intratumor deposition of chemotherapeutic agents. Here we investigated strategies to improve drug delivery to two tumor types that share in common poor drug delivery, but differ in the underlying cause. First, in an angiogenesis-driven brain tumor model of Glioblastoma, the vascular permeability barrier, along with poorly-functional vasculature, hinders drug delivery. A strategy of nanoparticle-based tumor 'priming' to attack the vascular permeability barrier, employing sterically stabilized liposomal doxorubicin (SSL-DXR), was investigated. Functional and histological evaluation of tumor vasculature revealed that after an initial period of depressed vascular permeability and vascular pruning 3--4 days after SSL-DXR administration, vascular permeability and perfusion were restored and then elevated after 5--7 days. As a result of tumor priming, deposition of subsequently-administered nanoparticles was enhanced, and the efficacy of temozolomide (TMZ), if administered during the window of elevated permeability, was increased. The sequenced regimen resulted in a persistent reduction of the tumor proliferative index and a 40% suppression of tumor volume, compared to animals that received both agents simultaneously. Second, in a hypovascular, pancreatic ductal adenocarcinoma model, disruption of tumor-stromal communication via sonic hedgehog (sHH) signaling pathway inhibition mediated an indirect vascular proliferation and a more than 2-fold increase in intratumor nanoparticle deposition. Enhanced delivery of SSL-DXR in tumors pre

  12. Formation of nucleoside 5'-polyphosphates from nucleotides and trimetaphosphate.

    PubMed

    Lohrmann, R

    1975-12-29

    When solutions of nucleoside 5'-phosphates and trimetaphosphate are dried out at room temperature, nucleoside 5'-polyphosphates are formed. The Mg++ ion shows a superior catalytic function in this reaction when compared with other divalent metal ions. Starting with nucleoside 5'-phosphates, Mg++ and trimetaphosphate, the predominant products in the nucleoside 5'-polyphosphate series pnN are p4N, P7N and p10N. Nucleoside 5'-diphosphates yield p5N and p8N, nucleoside 5'-triphosphates give p6N and p9N. The prebiological relevance of these reactions is discussed. PMID:1541

  13. Biosynthesis of riboflavin. An aliphatic intermediate in the formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphate.

    PubMed

    Neuberger, G; Bacher, A

    1985-02-28

    6,7-Dimethyl-8-ribityllumazine synthase deficient mutants of Candida guilliermondii were divided into two groups on the basis of in vitro complementation. Mutants of complementation group I produce an intermediate X from ribose 5-phosphate in a reaction requiring Mg++ ions. Compound X was partially purified and was shown to be a phosphoric acid ester. 6,7-Dimethyl-8-ribityllumazine can be formed from Compound X by cell extracts from mutants of complementation group II. The reaction requires 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate as second substrate. No divalent cations are required. PMID:3838473

  14. Antimutagenic effect of aqueous extract from Agaricus brasiliensis on culture of human lymphocytes.

    PubMed

    Gameiro, Paula H; Nascimento, José S; Rocha, Beatriz H G; Piana, Clause F B; Santos, Raquel A; Takahashi, Catarina S

    2013-02-01

    The mushroom Agaricus brasiliensis (sun mushroom), native from the southeast of Brazil, is well known by its medicinal properties that include effects on diabetes, cholesterol levels, and osteoporosis. The antimutagenic effects of A. brasiliensis has been investigated recently and revealed some controversial results depending on the temperature by which the A. brasiliensis tea is obtained. In the present study, we evaluated the effect of the A. brasiliensis extract prepared in two different temperatures, 4°C and 25°C, on the doxorubicin-induced DNA strand breaks and chromosomal aberrations (CAs) in human lymphocytes. The results demonstrated that A. brasiliensis was able to reduce the DXR-induced DNA damage in both temperatures; however, the CA test was more sensitive to demonstrate a better reduction when the cells were treated with an extract obtained at 25°C. A. brasiliensis extract obtained in different temperatures exhibited antigenotoxic and anticlastogenic effects in human lymphocytes.

  15. Design and synthesis of novel hydroxyanthraquinone nitrogen mustard derivatives as potential anticancer agents via a bioisostere approach.

    PubMed

    Zhao, Li-Ming; Ma, Feng-Yan; Jin, Hai-Shan; Zheng, Shilong; Zhong, Qiu; Wang, Guangdi

    2015-09-18

    A series of hydroxyanthraquinones having an alkylating N-mustard pharmacophore at 1'-position were synthesized via a bioisostere approach to evaluate their cytotoxicity against four tumor cell lines (MDA-MB-231, HeLa, MCF-7 and A549). These compounds displayed significant in vitro cytotoxicity against MDA-MB-231 and MCF-7 cells, reflecting the excellent selectivity for the human breast cancer. Among them, compound 5k was the most cytotoxic with IC50 value of 0.263 nM and is more potent than DXR (IC50 = 0.294 nM) in inhibiting the growth of MCF-7 cells. The excellent cytotoxicity and good selectivity of compound 5k suggest that it could be a promising lead for further design and development of anticancer agents, especially for breast cancer.

  16. Generation of coherent states of photon-added type via pathway of eigenfunctions

    NASA Astrophysics Data System (ADS)

    Górska, K.; Penson, K. A.; Duchamp, G. H. E.

    2010-09-01

    We obtain and investigate the regular eigenfunctions of simple differential operators xr dr + 1/dxr + 1, r = 1, 2, ..., with the eigenvalues equal to 1. With the help of these eigenfunctions, we construct a non-unitary analogue of a boson displacement operator which will be acting on the vacuum. In this way, we generate collective quantum states of the Fock space which are normalized and equipped with the resolution of unity with the positive weight functions that we obtain explicitly. These states are thus coherent states in the sense of Klauder. They span the truncated Fock space without first r lowest-lying basis states: |0rang, |1rang, ..., |r - 1rang. These states are squeezed, sub-Poissonian in nature and reminiscent of photon-added states in Agarwal and Tara (1991 Phys. Rev. A 43 492).

  17. [Complete resection for giant thymic carcinoma after simultaneous combination chemotherapy].

    PubMed

    Ueki, Tomoyuki; Ueshima, Y; Kurioka, H; Enoki, Y; Hosokawa, Y

    2005-04-01

    A 19-year-old man visited our hospital complaining of dyspnea. Chest X-ray and computed tomography (CT) showed a huge mass in the right anterior mediastinum. We diagnosed this as invasive thymoma by microscopic examination of specimens obtained by echo-guided needle biopsy. The patient underwent 6 courses chemotherapy [1st course : carboplatin (CBDCA) + doxorubicin hydrochloride (DXR) + vincristine sulfate (VCR) + cyclophosphamide (CPA), 2nd, 3rd-6th course : cisplatin (CDDP) + ADM + VCR + CPA]. At achievement of partial response (the reduction rate of the tumor size : 91.4%), the tumor was completely resected. The pathological examination of the resected specimens yielded a diagnosis of large cell carcinoma. Preoperative chemotherapy with ADOC regimen may be effective in advanced thymic carcinoma.

  18. Citral and eugenol modulate DNA damage and pro-inflammatory mediator genes in murine peritoneal macrophages.

    PubMed

    Porto, Marilia de Paula; da Silva, Glenda Nicioli; Luperini, Bruno Cesar Ottoboni; Bachiega, Tatiana Fernanda; de Castro Marcondes, João Paulo; Sforcin, José Maurício; Salvadori, Daisy Maria Fávero

    2014-11-01

    Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.

  19. Superoxide dismutase (SOD) genes in Streptomyces peucetius: effects of SODs on secondary metabolites production.

    PubMed

    Kanth, Bashistha Kumar; Jnawali, Hum Nath; Niraula, Narayan Prasad; Sohng, Jae Kyung

    2011-07-20

    Two superoxide dismutase (SOD) genes; sod1 and sod2, from Streptomyces peucetius ATCC 27952 show high similarity to other known SODs from Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680. These sod1 and sod2 were cloned into pIBR25 expression vector under a strong ermE* promoter to enhance secondary metabolites from Streptomyces strains. The recombinant expression plasmids; pIBR25SD1 and pIBR25SD2, were constructed to overexpress sod1 and sod2 respectively to enhance production of doxorubicin (DXR) in S. peucetius, clavulanic acid (CA) in Streptomyces clavuligerus NRRL 3585 and actinorhodin (ACT) and undecylprodigiosin (Red) in Streptomyces lividans TK24. Biomass variation, antibiotics production and transcriptional analysis of regulatory genes in recombinant strains have been studied to understand the effect of sod1 and sod2. The cell growth analysis shows that life span of all recombinant strains was found to be elevated as compared to wild type cells. In S. peucetius, overexpression of sod1 and sod2 was not effective in DXR production but in case of S. clavuligerus, CA production was increased by 2.5 and 1.5 times in sod1 and sod2 overexpression, respectively while in case of S. lividans, ACT production was increased by 1.4 and 1.6 times and Red production by 1.5 and 1.2 times upon sod1 and sod2 overexpressions, respectively as compared to the corresponding wild type strains. PMID:20888207

  20. The non-mevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria.

    PubMed

    Zeidler, J; Schwender, J; Mueller, C; Lichtenthaler, H K

    2000-12-01

    Two plant test systems are presented in the search for new inhibitors of the non-mevalonate isoprenoid pathway. A derivative of clomazone appears to be an inhibitor of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate (DOXP/MEP) pathway of isoprenoid formation.

  1. Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle

    SciTech Connect

    Panosian, Timothy D.; Nannemann, David P.; Watkins, Guy R.; Phelan, Vanessa V.; McDonald, W. Hayes; Wadzinski, Brian E.; Bachmann, Brian O.; Iverson, Tina M.

    2011-09-15

    Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert {alpha}-D-ribose 5-phosphate (ribose 5-phosphate) and {alpha}-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator {alpha}-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn{sup 2+}-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[{sup 18}O{sub 3}]phosphate and [U-{sup 13}C{sub 5}]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

  2. Bacillus subtilis GabR, a protein with DNA-binding and aminotransferase domains, is a PLP-dependent transcriptional regulator.

    PubMed

    Belitsky, Boris R

    2004-07-16

    Bacillus subtilis GabR is a member of a poorly characterized but widespread family of chimeric bacterial proteins that have apparent DNA binding and aminotransferase domains. GabR positively regulates expression of the gabTD operon responsible for utilization of gamma-aminobutyric acid (GABA) and represses the divergently transcribed gabR gene. Purified GabR bound specifically to the DNA region overlapping the -35 region of the gabT promoter and the -10 and +1 regions of the gabR promoter. Two 6 bp direct repeats located at the ends of this region appeared to be essential for GabR binding. In transcription reactions in vitro, GabR alone repressed expression from the gabR promoter but activated expression from the gabT promoter only in the presence of GABA and pyridoxal 5'-phosphate, an essential cofactor of aminotransferases. A similar requirement for pyridoxal 5'-phosphate and GABA for GabR-mediated transcription activation was shown in vivo. In vitro this requirement could be partially satisfied with pyridoxamine 5'-phosphate and succinic semialdehyde, the products of a GABA-dependent aminotransferase half-reaction. We hypothesize that the GabR-catalyzed aminotransferase-like reaction between GABA and pyridoxal 5'-phosphate is essential for GabR action as a transcriptional activator.

  3. Public health significance of elevated homocysteine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homocysteine is a sulfur amino acid whose metabolism stands at the intersection of two pathways: remethylation, which requires folic acid and vitamin B12 coenzymes; and transsulfuration, which requires pyridoxal-5'-phosphate, the vitamin B6 coenzyme. Data from a number of laboratories suggest that m...

  4. Plasma folate, vitamin B-6, vitamin B-12, and risk of breast cancer in women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: B vitamins such as folate, vitamin B-6, and vitamin B-12 are coenzymes that are important for DNA integrity and stability. Deficiency in these B vitamins may promote tumor carcinogenesis. Objective: We prospectively evaluated plasma concentrations of folate, pyridoxal 5'-phosphate (PLP; ...

  5. Unexpectedly facile synthesis of symmetrical P1,P2-dinucleoside-5'pyrophosphates

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia; Lu, Jonathan; Rosenbach, Morgan T.; Hurley, T. B.

    1991-01-01

    Symmetrical dinucleoside 5'-pyrophosphates have been synthesized from the corresponding nucleoside 5'-phosphate free acid in high yield. The one-pot procedure is carried out in DMF or DMSO using triphenylphosphine and 2,2'-dipyridyldisulfide as the coupling agents, and 1-methylimidazole as the catalyst.

  6. The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase.

    PubMed

    Sainis, J K; Merriam, K; Harris, G C

    1989-01-01

    When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl(2) followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.

  7. ENZYMATIC BASIS FOR D-ARBITOL PRODUCTION BY SACCHAROMYCES ROUXII.

    PubMed

    INGRAM, J M; WOOD, W A

    1965-05-01

    Ingram, Jordan M. (Michigan State University, East Lansing), and W. A. Wood. Enzymatic basis for d-arabitol production by Saccharomyces rouxii. J. Bacteriol. 89:1186-1194. 1965.-The enzymatic steps in d-arabitol synthesis by Saccharomyces rouxii were studied. The fermentation of d-glucose-6-C(14) gave rise to d-arabitol labeled at C-5; d-ribose of ribonucleic acid had the same isotope pattern. Crude extracts were able to reduce d-ribulose with reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) and d-xylulose with reduced nicotinamide adenine dinucleotide (NADH(2)). These extracts also oxidized d-arabitol with nicotinamide adenine dinucleotide phosphate and xylitol with nicotinamide adenine dinucleotide. No reduction of d-ribulose-5-phosphate or d-xylulose-5-phosphate was observed. An enzyme which reduced d-xylulose with NADH(2) was purified 33-fold and characterized as a xylitol (--> d-xylulose) dehydrogenase. Similarly, an enzyme reducing d-ribulose with NADPH(2) was purified 12-fold and characterized as a d-arabitol (--> d-ribulose) dehydrogenase. Alkaline and acid phosphatases were purified 50- and 40-fold, respectively, and their specificities were determined. Only the acid phosphatase had detectable activity on d-ribulose-5-phosphate. The data support the postulate that d-arabitol arises by dephosphorylation of d-ribulose-5-phosphate and reduction of d-ribulose by a NADPH(2)-linked d-arabitol (--> d-ribulose) dehydrogenase.

  8. Discernment of lint trash in raw cotton using multivariate analysis of excitation-emission luminescence spectra

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Excitation-Emission luminescence spectra of basic (pH 12.5) phosphate buffer solution extracts were used to distinguish among botanical components of trash within seed cotton. All components were separated from whole plants removed from a field in southern New Mexico. Unfolded Principal Component An...

  9. [Use of time-of-flight mass spectrometry with ionization division fragments of californium-252 for studying the mechanisms of action of drugs on DNA and its components].

    PubMed

    Sukhodub, L F; Grebenik, L I; Chivanov, V D

    1994-01-01

    Using soft-ionization mass spectrometry (252-Cf particle desorption mass spectrometry, PDMS) a minor adduct of anticancer drug prospidine and deoxyguanosine-5-phosphate (pdG) has been found. It has been shown experimentally that PDMS is very useful for study of biological mixtures as well as mechanisms of interactions between drugs and biomolecules.

  10. Prediagnostic plasma vitamin B6 (pyridoxal 50-phosphate) and survival in patients with colorectal cancer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Higher plasma pyridoxal 5'-phosphate (PLP) levels are associated with a decreased incidence of colorectal cancer, but the influence of plasma PLP on survival of patients with colorectal cancer is unknown. We prospectively examined whether prediagnostic plasma PLP levels are associated with mortality...

  11. Xylose utilization in recombinant zymomonas

    DOEpatents

    Caimi, Perry G; McCole, Laura; Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V

    2014-03-25

    Xylose-utilizing Zymomonas strains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

  12. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    SciTech Connect

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate

  13. The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

    PubMed Central

    MacLeod, John K.

    2006-01-01

    14C-Labelled octulose phosphates were formed during photosynthetic 14CO2 fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero-d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though 14C-labelled d-glycero-d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. 14CO2 predominantly labelled positions 5 and 6 of d-glycero-d-ido-octulose 1,8-P2 consistent with labelling predictions of the modified scheme. The kinetics of 14CO2 incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to 12CO2 rapidly displaced the 14C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO2.) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO2 assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO2 fixation at rates in excess of 120 μmol h−1 mg−1 Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP

  14. Transketolase A of Escherichia coli K12. Purification and properties of the enzyme from recombinant strains.

    PubMed

    Sprenger, G A; Schörken, U; Sprenger, G; Sahm, H

    1995-06-01

    Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145,000 Da (+/- 10,000 Da), judged by gel-filtration chromatography. Subunits of 73,000 Da (+/- 2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30 degrees C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 microM and 1.4 mM, respectively. Km values for the other physiological substrates of transketolase A were 90 microM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate. Hydroxypyruvate served as alternative donor (Km = 18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). The enzyme was competitively inhibited by D-arabinose 5-phosphate (K = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA

  15. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.

    PubMed

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

    There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme. PMID:22839733

  16. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    PubMed

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho

    2012-11-01

    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  17. New Insight into Isoprenoids Biosynthesis Process and Future Prospects for Drug Designing in Plasmodium

    PubMed Central

    Saggu, Gagandeep S.; Pala, Zarna R.; Garg, Shilpi; Saxena, Vishal

    2016-01-01

    The MEP (Methyl Erythritol Phosphate) isoprenoids biosynthesis pathway is an attractive drug target to combat malaria, due to its uniqueness and indispensability for the parasite. It is functional in the apicoplast of Plasmodium and its products get transported to the cytoplasm, where they participate in glycoprotein synthesis, electron transport chain, tRNA modification and several other biological processes. Several compounds have been tested against the enzymes involved in this pathway and amongst them Fosmidomycin, targeted against IspC (DXP reductoisomerase) enzyme and MMV008138 targeted against IspD enzyme have shown good anti-malarial activity in parasite cultures. Fosmidomycin is now-a-days prescribed clinically, however, less absorption, shorter half-life, and toxicity at higher doses, limits its use as an anti-malarial. The potential of other enzymes of the pathway as candidate drug targets has also been determined. This review details the various drug molecules tested against these targets with special emphasis to Plasmodium. We corroborate that MEP pathway functional within the apicoplast of Plasmodium is a major drug target, especially during erythrocytic stages. However, the major bottlenecks, bioavailability and toxicity of the new molecules needs to be addressed, before considering any new molecule as a potent antimalarial. PMID:27679614

  18. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.

    PubMed

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

    There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme.

  19. New Insight into Isoprenoids Biosynthesis Process and Future Prospects for Drug Designing in Plasmodium

    PubMed Central

    Saggu, Gagandeep S.; Pala, Zarna R.; Garg, Shilpi; Saxena, Vishal

    2016-01-01

    The MEP (Methyl Erythritol Phosphate) isoprenoids biosynthesis pathway is an attractive drug target to combat malaria, due to its uniqueness and indispensability for the parasite. It is functional in the apicoplast of Plasmodium and its products get transported to the cytoplasm, where they participate in glycoprotein synthesis, electron transport chain, tRNA modification and several other biological processes. Several compounds have been tested against the enzymes involved in this pathway and amongst them Fosmidomycin, targeted against IspC (DXP reductoisomerase) enzyme and MMV008138 targeted against IspD enzyme have shown good anti-malarial activity in parasite cultures. Fosmidomycin is now-a-days prescribed clinically, however, less absorption, shorter half-life, and toxicity at higher doses, limits its use as an anti-malarial. The potential of other enzymes of the pathway as candidate drug targets has also been determined. This review details the various drug molecules tested against these targets with special emphasis to Plasmodium. We corroborate that MEP pathway functional within the apicoplast of Plasmodium is a major drug target, especially during erythrocytic stages. However, the major bottlenecks, bioavailability and toxicity of the new molecules needs to be addressed, before considering any new molecule as a potent antimalarial.

  20. FgIlv5 is required for branched-chain amino acid biosynthesis and full virulence in Fusarium graminearum.

    PubMed

    Liu, Xin; Wang, Jian; Xu, Jianhong; Shi, Jianrong

    2014-04-01

    In this study, we characterized FgIlv5, a homologue of the Saccharomyces cerevisiae keto-acid reductoisomerase (KARI) from the important wheat head scab fungus Fusarium graminearum. KARI is a key enzyme in the branched-chain amino acid (BCAA, including leucine, isoleucine and valine) biosynthetic pathway that exists in a variety of organisms from bacteria to fungi and higher plants, but not in mammals. The FgILV5 deletion mutant ΔFgIlv5-4 failed to grow when the culture medium was nutritionally limited for BCAAs. When grown on potato-dextrose agar plates, ΔFgIlv5-4 exhibited a significant decrease in aerial hyphae formation and red pigmentation. Conidia formation was also blocked in ΔFgIlv5-4. Exogenous addition of 1 mM isoleucine and valine was able to rescue the defects of mycelial growth and conidial morphogenesis. Cellular stress assays showed that ΔFgIlv5-4 was more sensitive to osmotic and oxidative stresses than the wild-type strain. In addition, virulence of ΔFgIlv5-4 was dramatically reduced on wheat heads, and a low level of deoxynivalenol production was detected in ΔFgIlv5-4 in wheat kernels. The results of this study indicate that FgIlv5 is involved in valine and isoleucine biosynthesis and is required for full virulence in F. graminearum.

  1. Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

    PubMed Central

    Wang, Ju; Lin, Jianxun; Zhao, Minyan

    2015-01-01

    Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO. PMID:25815733

  2. Proteomic analysis of cell wall in four pathogenic species of Candida exposed to oxidative stress.

    PubMed

    Ramírez-Quijas, Mayra Denisse; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2015-10-01

    In order for Candida species to adhere and colonize human host cells they must express cell wall proteins (CWP) and adapt to reactive oxygen species (ROS) generated by phagocytic cells of the human host during the respiratory burst. However, how these pathogens change the expression of CWP in response to oxidative stress (OSR) is not known. Here, fifteen moonlight-like CWP were identified that expressed differentially in four species of Candida after they were exposed to H2O2 or menadione (O2(-)). These proteins included: (i) glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), fructose-bisphosphate aldolase (Fba1), phosphoglycerate mutase (Gpm1), phosphoglycerate kinase (Pgk), pyruvate kinase (Pk) and enolase (Eno1); (ii) the heat shock proteins Ssb1 and Ssa2; (iii) OSR proteins such as peroxyredoxin (Tsa1), the stress protein Ddr48 (Ddr48) and glutathione reductase (Glr1); (iv) other metabolic enzymes such as ketol-acid reductoisomerase (Ilv5) and pyruvate decarboxylase (Pdc1); and (v) other proteins such as elongation factor 1-beta (Efb1) and the 14-3-3 protein homolog. RT-PCR revealed that transcription of the genes coding for some of the identified CWP are differentially regulated. To our knowledge this is the first report showing that moonlight-like CWP are the first line of defense of Candida against ROS, and that they are differentially regulated in each of these pathogens.

  3. New Insight into Isoprenoids Biosynthesis Process and Future Prospects for Drug Designing in Plasmodium.

    PubMed

    Saggu, Gagandeep S; Pala, Zarna R; Garg, Shilpi; Saxena, Vishal

    2016-01-01

    The MEP (Methyl Erythritol Phosphate) isoprenoids biosynthesis pathway is an attractive drug target to combat malaria, due to its uniqueness and indispensability for the parasite. It is functional in the apicoplast of Plasmodium and its products get transported to the cytoplasm, where they participate in glycoprotein synthesis, electron transport chain, tRNA modification and several other biological processes. Several compounds have been tested against the enzymes involved in this pathway and amongst them Fosmidomycin, targeted against IspC (DXP reductoisomerase) enzyme and MMV008138 targeted against IspD enzyme have shown good anti-malarial activity in parasite cultures. Fosmidomycin is now-a-days prescribed clinically, however, less absorption, shorter half-life, and toxicity at higher doses, limits its use as an anti-malarial. The potential of other enzymes of the pathway as candidate drug targets has also been determined. This review details the various drug molecules tested against these targets with special emphasis to Plasmodium. We corroborate that MEP pathway functional within the apicoplast of Plasmodium is a major drug target, especially during erythrocytic stages. However, the major bottlenecks, bioavailability and toxicity of the new molecules needs to be addressed, before considering any new molecule as a potent antimalarial. PMID:27679614

  4. Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Adem, Şevki; Kayhan, Naciye

    2016-04-01

    6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

  5. Light/dark modulation of enzyme activity in developing barley leaves

    SciTech Connect

    Sibley, M.H.; Anderson, L.E. )

    1989-12-01

    Light/dark modulation of the ribulose-5-phosphate kinase, NADP{sup +}-glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase activity was measured in the developing primary leaf of barley (Hordeum vulgare L.) seedlings. Ribulose-5-phosphate kinase and NADP{sup +}-glyceraldehyde-3-phosphate dehydrogenase were fully light activated even at the earliest developmental stage sampled. In contrast, light modulation of fructose-1,6-bisphosphatase exhibited a complex response to leaf developmental status. Light stimulation of fructose-1,6-bisphosphatase activity (measured at pH 8.0) increased progressively during leaf development. On the other hand, acid fructose-1,6-bisphosphatase activity (measured at pH 6.0) was inhibited by light, and this light inhibition was greater in the base of the leaf than in the tip of the leaf.

  6. Biosynthesis of riboflavin. Studies on the mechanism of L-3,4-dihydroxy-2-butanone 4-phosphate synthase.

    PubMed

    Volk, R; Bacher, A

    1991-11-01

    The riboflavin precursor, L-3,4-dihydroxy-2-butanone 4-phosphate, is formed from D-ribulose 5-phosphate by a single 24-kDa enzyme. Studies with various specifically 13C-labeled D-ribulose 5-phosphates as substrate showed that the carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-5 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-5 of the substrate is an intramolecular reaction. The hydrogen atom at C-3 of the enzyme product is introduced from solvent water. A reaction mechanism which is in agreement with all experimental data is proposed. PMID:1939111

  7. ATP requirements and small interfering RNA structure in the RNA interference pathway.

    PubMed

    Nykänen, A; Haley, B; Zamore, P D

    2001-11-01

    We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

  8. Photosynthesis in Rhodospirillum rubrum. III. Metabolic Control of Reductive Pentose Phosphate and Tricarboxylic Acid Cycle Enzymes 1

    PubMed Central

    Anderson, Louise; Fuller, R. C.

    1967-01-01

    Enzymes of the reductive pentose phosphate cycle including ribulose-diphosphate carboxylase, ribulose-5-phosphate kinase, ribose-5-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose-1,6-diphos-phatase were shown to be present in autotrophically grown Rhodospirillum rubrum. Enzyme levels were measured in this organism grown photo- and dark heterotrophically as well. Several, but not all, of these enzymes appeared to be under metabolic control, mediated by exogenous carbon and nitrogen compounds. Light had no effect on the presence or levels of any of these enzymes in this photosynthetic bacterium. The enzymes of the tricarboxylic acid cycle and enolase were shown to be present in R. rubrum cultured aerobically, autotrophically, or photoheterotrophically, both in cultures evolving hydrogen and under conditions where hydrogen evolution is not observed. Light had no clearly demonstrable effect on the presence or levels of any of these enzymes. PMID:6042359

  9. The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

    SciTech Connect

    Diaz, Z.; Xavier, K; Miller, S

    2009-01-01

    Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (??)8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology with aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase.

  10. Vitamin B6 biosynthesis in higher plants

    PubMed Central

    Tambasco-Studart, Marina; Titiz, Olca; Raschle, Thomas; Forster, Gabriela; Amrhein, Nikolaus; Fitzpatrick, Teresa B.

    2005-01-01

    Vitamin B6 is an essential metabolite in all organisms. It can act as a coenzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have a de novo biosynthetic pathway for vitamin B6, but animals must obtain it from dietary sources. In Escherichia coli, it is known that the vitamin is derived from deoxyxylulose 5-phosphate (an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis) and 4-phosphohydroxy-l-threonine. It has been assumed that vitamin B6 is synthesized in the same way in plants, but this hypothesis has never been experimentally proven. Here, we show that, in plants, synthesis of the vitamin takes an entirely different route, which does not involve deoxyxylulose 5-phosphate but instead utilizes intermediates from the pentose phosphate pathway, i.e., ribose 5-phosphate or ribulose 5-phosphate, and from glycolysis, i.e., dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. The revelation is based on the recent discovery that, in bacteria and fungi, a novel pathway is in place that involves two genes (PDX1 and PDX2), neither of which is homologous to any of those involved in the previously doctrined E. coli pathway. We demonstrate that Arabidopsis thaliana has two functional homologs of PDX1 and a single homolog of PDX2. Furthermore, and contrary to what was inferred previously, we show that the pathway appears to be cytosolic and is not localized to the plastid. Last, we report that the single PDX2 homolog is essential for plant viability. PMID:16157873

  11. [Effect of aggregating agents on the pentosephosphate pathway of carbohydrate metabolism in human blood platelet extracts].

    PubMed

    Makarov, S A; Kudriavtseva, G V; Kolotilova, A I

    1983-01-01

    Thrombin inhibits the rate of glucose-6-phosphate and 6-phosphogluconate oxidation. ADP decreases the incorporation of ribose-5-phosphate into metabolism. The effect of adrenaline on the pentose phosphate pathway reactions was not demonstrated. Cell destruction by means of freezing, thawing and treatment with Triton X-100 decreases the rate of the glucose-6-phosphate dehydrogenase reaction to a greater degree as compared with osmotic shock.

  12. [The pentose phosphate pathway and NADP-dependent glycerol-3-phosphate dehydrogenase activity in some tissues of albino rat].

    PubMed

    Glushankov, E P; Epifanova, Iu E; Kolotilova, A I

    1976-10-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity in liver, heart and skeletal muscle of rat was studied. The activity is found when glyceraldehyde-3-phosphate or ribose-5-phosphate in the presence of ATP are taken as substrates. The data obtained confirm that NADP-dependent glycerol-3-phosphate dehydrogenase exists in skeletal muscle and demonstrate that it is found in heart muscle as well.

  13. 4'-CyanoPLP presents better prospect for the experimental detection of elusive cyclic intermediate radical in the reaction of lysine 5,6-aminomutase.

    PubMed

    Maity, Amarendra Nath; Ke, Shyue-Chu

    2015-02-01

    The results of our calculations suggest that the reaction of 4'-cyanoPLP with lysine 5,6-aminomutase offers better prospect for the experimental detection of elusive cyclic azacyclopropylcarbinyl radical (I), which is proposed to be a key intermediate in the reaction of pyridoxal-5'-phosphate dependent radical aminomutases. We have calculated the corresponding hyperfine coupling constants (HFCCs) for (14)N and (13)C of cyano group using several basis sets to help the characterization of 4'-cyanoI.

  14. Structure of Escherichia coli tryptophanase purified from an alkaline-stressed bacterial culture.

    PubMed

    Rety, Stephane; Deschamps, Patrick; Leulliot, Nicolas

    2015-11-01

    Tryptophanase is a bacterial enzyme involved in the degradation of tryptophan to indole, pyruvate and ammonia, which are compounds that are essential for bacterial survival. Tryptophanase is often overexpressed in stressed cultures. Large amounts of endogenous tryptophanase were purified from Escherichia coli BL21 strain overexpressing another recombinant protein. Tryptophanase was crystallized in space group P6522 in the apo form without pyridoxal 5'-phosphate bound in the active site.

  15. 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.

  16. Pnp gene modification for improved xylose utilization in Zymomonas

    DOEpatents

    Caimi, Perry G G; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

    2014-12-16

    The endogenous pnp gene encoding polynucleotide phosphorylase in the Zymomonas genome was identified as a target for modification to provide improved xylose utilizing cells for ethanol production. The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification, and are not limited in xylose isomerase activity in the absence of the pnp modification.

  17. Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

    SciTech Connect

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

    1988-08-09

    The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30 a shows a carbon isotope effect k/sup 12//k/sup 13/ = 1.0334 +/- 0.0005 and a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9799 +/- 0.0006 at pH 4.8, 37/sup 0/C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D/sub 2/O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.

  18. Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

    SciTech Connect

    Perez-Ramirez, B.; Martinez-Carrion, M. )

    1989-06-13

    A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with ({sup 3}H)pyridoxine 5-phosphate (({sup 3}H)PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of ({sup 3}H)PNP at 37{degree}C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the {alpha}-subunit, internal surface labeling by PLP was greatest for the highest molecular weight ({delta}) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles.

  19. Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes.

    PubMed Central

    Connolly, B A; Rider, P

    1985-01-01

    Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derivatised with thiol specific probes. The nucleotide sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed using the S-triphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (1) ol or 6-mercaptohexan (1) ol. After cleavage from the resin and removal of the phosphate and base protecting groups, this yields an oligonucleotide containing an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three or six carbon chain. The triphenylmethyl group can be readily removed with silver nitrate to give the free thiol. With the three and six carbon chain oligonucleotides, this thiol can be used, at pH 8, for the attachment of thiol specific probes as illustrated by the reaction with fluorescent conjugates of iodoacetates and maleiimides. However, oligonucleotides containing a thiol attached to the 5'-phosphate group via a two carbon chain are unstable at pH 8 decomposing to the free 5'-phosphate and so are unsuitable for further derivatisation. PMID:4011448

  20. Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

    PubMed

    Börner, Tim; Grey, Carl; Adlercreutz, Patrick

    2016-08-01

    Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.

  1. Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release.

    PubMed

    Spégel, Peter; Sharoyko, Vladimir V; Goehring, Isabel; Danielsson, Anders P H; Malmgren, Siri; Nagorny, Cecilia L F; Andersson, Lotta E; Koeck, Thomas; Sharp, Geoffrey W G; Straub, Susanne G; Wollheim, Claes B; Mulder, Hindrik

    2013-03-15

    Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

  2. A novel approach to inhibit intracellular vitamin B6-dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase.

    PubMed

    Wu, Fang; Christen, Philipp; Gehring, Heinz

    2011-07-01

    Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 μM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest. PMID:21454364

  3. Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release.

    PubMed

    Spégel, Peter; Sharoyko, Vladimir V; Goehring, Isabel; Danielsson, Anders P H; Malmgren, Siri; Nagorny, Cecilia L F; Andersson, Lotta E; Koeck, Thomas; Sharp, Geoffrey W G; Straub, Susanne G; Wollheim, Claes B; Mulder, Hindrik

    2013-03-15

    Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling. PMID:23282133

  4. Microbial growth on C1 compounds. Incorporation of C1 units into allulose phosphate by extracts of Pseudomonas methanica

    PubMed Central

    Kemp, M. B.; Quayle, J. R.

    1966-01-01

    1. Incubation of cell-free extracts of methane- or methanol-grown Pseudomonas methanica with [14C]formaldehyde and d-ribose 5-phosphate leads to incorporation of radioactivity into a non-volatile product, which has the chromatographic properties of a phosphorylated compound. 2. Treatment of this reaction product with a phosphatase, followed by chromatography, shows the presence of two compounds whose chromatographic properties are consistent with their being free sugars. 3. The minor component of the dephosphorylated products has been identified as fructose. The major component has been identified as allulose (psicose) on the basis of co-chromatography, co-crystallization of the derived phenylosazone and dinitrophenylosazone with authentic derivatives of allulose and behaviour towards oxidation with bromine water. 4. It is suggested that the bacterial extracts catalyse the condensation of a C1 unit identical with, or derived from, formaldehyde with ribose 5-phosphate to give allulose 6-phosphate. 5. Testing of hexose phosphates and pentose phosphates as substrates has so far shown the reaction to be specific for ribose 5-phosphate. 6. The condensation reaction is not catalysed by extracts of methanol-grown Pseudomonas AM1. 7. A variant of the pentose phosphate cycle, involving this condensation reaction, is suggested as an explanation for the net synthesis of C3 compounds from C1 units by P. methanica. PMID:5965346

  5. Human ISPD Is a Cytidyltransferase Required for Dystroglycan O-Mannosylation.

    PubMed

    Riemersma, Moniek; Froese, D Sean; van Tol, Walinka; Engelke, Udo F; Kopec, Jolanta; van Scherpenzeel, Monique; Ashikov, Angel; Krojer, Tobias; von Delft, Frank; Tessari, Marco; Buczkowska, Anna; Swiezewska, Ewa; Jae, Lucas T; Brummelkamp, Thijn R; Manya, Hiroshi; Endo, Tamao; van Bokhoven, Hans; Yue, Wyatt W; Lefeber, Dirk J

    2015-12-17

    A unique, unsolved O-mannosyl glycan on α-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional α-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.

  6. Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis.

    PubMed

    Colinas, Maite; Eisenhut, Marion; Tohge, Takayuki; Pesquera, Marta; Fernie, Alisdair R; Weber, Andreas P M; Fitzpatrick, Teresa B

    2016-02-01

    Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5'-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5'-phosphate (PMP), pyridoxine 5'-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism. PMID:26858304

  7. Interaction between vitamin B6 metabolism, nitrogen metabolism and autoimmunity.

    PubMed

    Colinas, Maite; Fitzpatrick, Teresa B

    2016-01-01

    The essential micronutrient vitamin B6 is best known in its enzymatic cofactor form, pyridoxal 5'-phosphate (PLP). However, vitamin B6 comprises the amine pyridoxamine 5'-phosphate (PMP) and the alcohol pyridoxine 5'-phosphate (PNP) in addition to PLP, as well as their corresponding non-phosphorylated forms. The different B6 forms (called vitamers) are enzymatically interconverted in a ubiquitous salvage pathway. Recently, we have shown that balancing the ratio of the different B6 vitamers in particular PMP by the PMP/PNP oxidase PDX3 is essential for growth and development in Arabidopsis thaliana. Intriguingly, nitrate to ammonium conversion is impaired in pdx3 mutants, such that the mutants become ammonium-dependent, suggesting an interaction between vitamin B6 and nitrogen metabolism. In addition, we found a strong up-regulation of genes related to plant defense. Here, we further show that pdx3 mutants display a temperature-sensitive phenotype that is typical of autoimmune mutants and is possibly connected to the impaired nitrogen metabolism. PMID:27018849

  8. Pyridoxine supply in human development.

    PubMed

    Bowling, Francis Gerard

    2011-08-01

    Vitamin B(6) has an important role in the function of the human nervous system. Experimental data are not generally available on the role in human development, but significant conclusions may be made from studies of the effect of disorders of B(6) vitamer metabolism. Vitamin B(6) comprises seven compounds - pyridoxal, pyridoxine, pyridoxamine and their respective 5' phosphates. The common active form in human tissue is the 5'-phosphate form of pyridoxal (PLP) most of which is found in muscle bound to phosphorylase. Like many vitamins, B(6) can function both as a co-enzyme and as a chaperone. Pyridoxal-5'-phosphate is the metabolically active form and is involved in 100 enzymatic reactions including carbohydrate, amino acid, and fatty acid metabolism. There is evidence that in some situations B(6) vitamers can function as antioxidants. The fetus is dependent on the placenta for supply of vitamin B(6) and the demand correlates with amino acid metabolism. Few reports are available on the role of B(6) in embryogenesis. Studies of human disorders where B(6) metabolism is blocked show a major role in neurotransmitter function with secondary cerebral and cerebellar hypoplasia. Pyridoxine potentiates vitamin A teratogenicity and an excess leads to peripheral nerve cell degeneration. The key role of vitamin B(6) in the developing human is in metabolism, especially of the neurotransmitters.

  9. Recombinogenic activity of Pantoprazole(®) in somatic cells of Drosophila melanogaster.

    PubMed

    Lopes, Jeyson Césary; Machado, Nayane Moreira; Saturnino, Rosiane Soares; Nepomuceno, Júlio César

    2015-03-01

    Pantoprazole(®) is one of the leading proton pump inhibitors (PPIs) used in the treatment of a variety of diseases related to the upper gastrointestinal tract. However, studies have shown an increased risk of developing gastric cancer, intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In the present study, the somatic mutation and recombination test (SMART) was employed to determine the mutagenic effects of Pantoprazole on Drosophila melanogaster. Repeated treatments with Pantoprazole were performed on 72-hour larvae of the standard (ST) and high bioactivation (HB) crosses at concentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) was administered at 0.4 mM, as a positive control. When administered to ST descendants, total number of spots were statistically significant at 2.5 and 5.0 μM concentrations. For HB descendants, a significant increase in the total number of spots was observed among the marked transheterozygous (MH) flies. Through analysis of balancer heterozygous (BH) descendants, recombinogenic effects were observed at all concentrations in descendants of the HB cross. In view of these experimental conditions and results, it was concluded that Pantoprazole is associated with recombinogenic effects in Drosophila melanogaster. PMID:25983631

  10. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

    PubMed

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

    2015-09-22

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

  11. Mitochondrial DNA is a direct target of anti-cancer anthracycline drugs

    SciTech Connect

    Ashley, Neil Poulton, Joanna

    2009-01-16

    The anthracyclines, such as doxorubicin (DXR), are potent anti-cancer drugs but they are limited by their clinical toxicity. The mechanisms involved remain poorly understood partly because of the difficulty in determining sub-cellular drug localisation. Using a novel method utilising the fluorescent DNA dye PicoGreen, we found that anthracyclines intercalated not only into nuclear DNA but also mitochondrial DNA (mtDNA). Intercalation of mtDNA by anthracyclines may thus contribute to the marked mitochondrial toxicity associated with these drugs. By contrast, ethidium bromide intercalated exclusively into mtDNA, without interacting with nuclear DNA, thereby explaining why mtDNA is the main target for ethidium. By exploiting PicoGreen quenching we also developed a novel assay for quantification of mtDNA levels by flow-cytometry, an approach which should be useful for studies of mitochondrial dysfunction. In summary our PicoGreen assay should be useful to study drug/DNA interactions within live cells, and facilitate therapeutic drug monitoring and kinetic studies in cancer patients.

  12. Synthesis and properties of a newly obtained sorbent based on silica gel coated with a polyaniline film as the stationary phase for non-suppressed ion chromatography.

    PubMed

    Sowa, Ireneusz; Wójciak-Kosior, Magdalena; Drączkowski, Piotr; Strzemski, Maciej; Kocjan, Ryszard

    2013-07-17

    The new sorbent for non-suppressed ion chromatography based on silica gel coated with a film of polyaniline (PANI) was obtained in a process of in situ polymerization of aniline by oxidation with ammonium peroxydisulfate. Raman analyses performed using a Thermo Scientific DXR confocal Raman Microscope equipped with the Omnic 8 software from Thermo Fisher Scientific have proved a uniform distribution of PANI on the surface of chromatographic beads and in the pores of the particle. The obtained stationary phase was packed into a stainless steel HPLC column. The quality of the prepared column was verified on the basis of hydrodynamic parameters such as column efficiency index (Ip) and separation impedance (E). The potential application of silica gel modified with polyaniline for separation and determination of selected anions using non-suppressed ion chromatography was also investigated. Chromatographic analyses were performed using of the ion chromatograph Dionex DX-500. The best results were obtained for the mobile phase consisting of an aqueous or methanolic solution of hydrochloric acid. The tested column and optimized chromatographic system were successfully used for the analysis of nitrate, bromide and iodide in waters samples.

  13. Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient.

    PubMed

    Fritze, Andreas; Hens, Felicitas; Kimpfler, Andrea; Schubert, Rolf; Peschka-Süss, Regine

    2006-10-01

    This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependent drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 degrees C or 30% at 37 degrees C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.

  14. Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera

    PubMed Central

    Singh, Gaurav; Tiwari, Manish; Singh, Surendra Pratap; Singh, Surendra; Trivedi, Prabodh Kumar; Misra, Pratibha

    2016-01-01

    Sterol glycosyltransferases (SGTs) catalyse transfer of glycon moiety to sterols and their related compounds to produce diverse glyco-conjugates or steryl glycosides with different biological and pharmacological activities. Functional studies of SGTs from Withania somnifera indicated their role in abiotic stresses but details about role under biotic stress are still unknown. Here, we have elucidated the function of SGTs by silencing SGTL1, SGTL2 and SGTL4 in Withania somnifera. Down-regulation of SGTs by artificial miRNAs led to the enhanced accumulation of withanolide A, withaferin A, sitosterol, stigmasterol and decreased content of withanoside V in Virus Induced Gene Silencing (VIGS) lines. This was further correlated with increased expression of WsHMGR, WsDXR, WsFPPS, WsCYP710A1, WsSTE1 and WsDWF5 genes, involved in withanolide biosynthesis. These variations of withanolide concentrations in silenced lines resulted in pathogen susceptibility as compared to control plants. The infection of Alternaria alternata causes increased salicylic acid, callose deposition, superoxide dismutase and H2O2 in aMIR-VIGS lines. The expression of biotic stress related genes, namely, WsPR1, WsDFS, WsSPI and WsPR10 were also enhanced in aMIR-VIGS lines in time dependent manner. Taken together, our observations revealed that a positive feedback regulation of withanolide biosynthesis occurred by silencing of SGTLs which resulted in reduced biotic tolerance. PMID:27146059

  15. Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites.

    PubMed

    Khadka, Manoj; Salem, Mohamed; Leblond, Jeffrey D

    2015-01-01

    Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3β-ol, and a minor unknown sterol which appeared to be a C(28:4) compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway (MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate (MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes (dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted.

  16. Hordeum chilense genome, a useful tool to investigate the endosperm yellow pigment content in the Triticeae

    PubMed Central

    2012-01-01

    Background The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae. Results Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2Hch, 3Hch and 7Hch in H. chilense, the former being the most significant one. Conclusions The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2Hch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the Hch genome may constitute a valuable tool for YPC studies in the Triticeae. PMID:23122232

  17. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

    PubMed Central

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W.; Ben Mamoun, Choukri; Altman, Sidney

    2015-01-01

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome. PMID:26351679

  18. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

    PubMed

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

    2015-09-22

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome. PMID:26351679

  19. Archaeometric studies by neutron, x-ray radiography and microCT

    NASA Astrophysics Data System (ADS)

    Latini, R. M.; Bellido, A. V. B.; Vinagre Filho, U. M.; Souza, M. I. S.; Lima, I.; Oliveira, D. F.; Lopes, R. T.

    2013-05-01

    The aim of this study is to investigate manufacturing techniques used in prehistoric Brazilian pottery from Acre state and Araruama, Rio de Janeiro state, Brazil, using Neutron and X-Ray Radiography. For the neutrongraphy different fragments of pottery were submitted to a neutron flux of the order of 105n.cm-2.s-1 for 3 minutes at the Argonauta research reactor of the Instituto de Engenharia Nuclear (IEN)/CNEN. Digital processing techniques using imaging plate were applied to process the image of the selected sample. For the radiography the sample were exposed to an X-Rays in the Feinfocus Model FX100 and the image was obtained by Flat Panel GE IT Model DXR 250V at the Laboratório de Instrumentação Nuclear (LIN) - COPPE/UFRJ. The Neutrongraphy and radiography shows two different manufacturing details: palette and rollers and the microtomography shows cavities in the clay body and different temper applied in the pottery production. The preliminary results shows promising techniques applied for the pottery manufacturing information and as complement for better understanding the ceramics classification and precedence.

  20. Development of a single aptamer-based surface enhanced Raman scattering method for rapid detection of multiple pesticides.

    PubMed

    Pang, Shintaro; Labuza, Theodore P; He, Lili

    2014-04-21

    The objective of this study was to develop a simple and rapid method that could detect and discriminate four specific pesticides (isocarbophos, omethoate, phorate, and profenofos) using a single aptamer-based capture procedure followed by Surface Enhanced Raman Spectroscopy (SERS). The aptamer is a single stranded DNA sequence that is specific to capture these four pesticides. The thiolated aptamer was conjugated onto silver (Ag) dendrites, a nanostructure that can enhance the Raman fingerprint of pesticides, through Ag-thiol bonds. It was then backfilled with 6-mercaptohexanol (MH) to prevent nonspecific binding. The modified SERS platform [Ag-(Ap + MH)] was then mixed with each pesticide solution (P) for 20 min. After capturing the pesticides, the Ag-(Ap + MH)-P complex was analyzed under a DXR Raman microscope and TQ Analyst software. The results show that the four pesticides can be captured and detected using principal component analysis based on their distinct fingerprint Raman peaks. The limits of detection (LODs) of isocarbophos, omethoate, phorate, and profenofos were 3.4 μM (1 ppm), 24 μM (5 ppm), 0.4 μM (0.1 ppm), and 14 μM (5 ppm) respectively. This method was also validated successfully in apple juice. These results demonstrated the super capacity of aptamer-based SERS in rapid detection and discrimination of multi-pesticides. This technique can be extended to detect a wide range of pesticides using specific aptamers. PMID:24551875

  1. Recombinogenic activity of Pantoprazole® in somatic cells of Drosophila melanogaster

    PubMed Central

    Lopes, Jeyson Césary; Machado, Nayane Moreira; Saturnino, Rosiane Soares; Nepomuceno, Júlio César

    2015-01-01

    Pantoprazole® is one of the leading proton pump inhibitors (PPIs) used in the treatment of a variety of diseases related to the upper gastrointestinal tract. However, studies have shown an increased risk of developing gastric cancer, intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In the present study, the somatic mutation and recombination test (SMART) was employed to determine the mutagenic effects of Pantoprazole on Drosophila melanogaster. Repeated treatments with Pantoprazole were performed on 72-hour larvae of the standard (ST) and high bioactivation (HB) crosses at concentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) was administered at 0.4 mM, as a positive control. When administered to ST descendants, total number of spots were statistically significant at 2.5 and 5.0 μM concentrations. For HB descendants, a significant increase in the total number of spots was observed among the marked transheterozygous (MH) flies. Through analysis of balancer heterozygous (BH) descendants, recombinogenic effects were observed at all concentrations in descendants of the HB cross. In view of these experimental conditions and results, it was concluded that Pantoprazole is associated with recombinogenic effects in Drosophila melanogaster. PMID:25983631

  2. Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose.

    PubMed

    Klutts, J Stacey; Hatanaka, Kenichi; Pan, Y T; Elbein, Alan D

    2002-02-15

    d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and

  3. Genome tailoring powered production of isobutanol in continuous CO2/H2 blend fermentation using engineered acetogen biocatalyst.

    PubMed

    Gak, Eugene; Tyurin, Michael; Kiriukhin, Michael

    2014-05-01

    The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta-ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min (p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min (p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO2/H2 gas blend. Biocatalyst Clostridium sp. MT871RG- 11IBR6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min (p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO2/H2 blend thus contributing to the reversal of global warming.

  4. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.

    PubMed

    Stincone, Anna; Prigione, Alessandro; Cramer, Thorsten; Wamelink, Mirjam M C; Campbell, Kate; Cheung, Eric; Olin-Sandoval, Viridiana; Grüning, Nana-Maria; Krüger, Antje; Tauqeer Alam, Mohammad; Keller, Markus A; Breitenbach, Michael; Brindle, Kevin M; Rabinowitz, Joshua D; Ralser, Markus

    2015-08-01

    The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner-Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the 'Warburg effect' of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite

  5. Effect of Ultraviolet A-induced Crosslinking on Dentin Collagen Matrix

    PubMed Central

    Seseogullari-Dirihan, Roda; Tjäderhane, Leo; Pashley, David H; Tezvergil-Mutluay, Arzu

    2016-01-01

    Objectives The aim of this study was to evaluate the effect of using UVA-induced crosslinking with or without riboflavin as photosensitizers on degradation of dentin matrix by dentin proteases. Methods Demineralized dentin specimens (0.4×3×6mm, n=10/group) were subjected to: (RP1), 0.1% riboflavin-5 phosphate/UVA for 1 min; (RP5), 0.1% riboflavin-5 phosphate/UVA for 5 min; (R1), 0.1% riboflavin/UVA for 1 min; (R5), 0.1% riboflavin-UVA for 5 min; (UV1), UVA for 1 min; (UV5), UVA for 5 min. Specimens were incubated in 1 mL zinc and calcium containing media for 1 day and 1 week. An untreated group served as control (CM). After incubation, the loss of dry mass of samples was measured and aliquots of media were analyzed for the release of C-terminal fragment telopeptide (ICTP vs CTX) of collagen to evaluate for cathepsin K (CA-K) and total matrix metalloproteinase (MMP)-mediated degradation. Data were analyzed using repeated measures ANOVA at α=0.05. Results Although UVA radiation alone reduced dentin degradation, UVA-activated riboflavin or riboflavin-5 phosphate inhibited MMP and CA-K activities more than UVA alone. The effects of crosslinking were more pronounced in 7-day samples; only with CA-K were the effects of crosslinking with or without photosensitizer significantly different from controls in 1-day samples. Significance The use of bioactive forms (RP) or longer treatment time did not result with better effect. The use of UVA crosslinking reduces dentin matrix degradation, especially with photosensitizers. PMID:26314255

  6. Mutations in the yeast LCB1 and LCB2 genes, including those corresponding to the hereditary sensory neuropathy type I mutations, dominantly inactivate serine palmitoyltransferase.

    PubMed

    Gable, Ken; Han, Gongshe; Monaghan, Erin; Bacikova, Dagmar; Natarajan, Mukil; Williams, Robert; Dunn, Teresa M

    2002-03-22

    It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p. PMID:11781309

  7. Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates.

    PubMed

    Nielsen, P; Neuberger, G; Fujii, I; Bown, D H; Keller, P J; Floss, H G; Bacher, A

    1986-03-15

    The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies. PMID:3949782

  8. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

    PubMed Central

    Stincone, Anna; Prigione, Alessandro; Cramer, Thorsten; Wamelink, Mirjam M. C.; Campbell, Kate; Cheung, Eric; Olin-Sandoval, Viridiana; Grüning, Nana-Maria; Krüger, Antje; Alam, Mohammad Tauqeer; Keller, Markus A.; Breitenbach, Michael; Brindle, Kevin M.; Rabinowitz, Joshua D.; Ralser, Markus

    2015-01-01

    The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and

  9. 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

  10. [Adrenaline and cyclic AMP stimulation of ketopentose and sedoheptulose formation in rat liver homogenates].

    PubMed

    Kolotilova, A I; Glushankov, E P; Epifanova, Iu E

    1976-01-01

    Formation of sedoheptulose-7-phosphate and ketopentose phosphate was studied in vitro as affected by epinephrine and cAMP. No effect of epinephrine on the activity of transketolase was found with ribose-5-phosphate as a substrate of the nonoxidative reactions of the pentose phosphate ccyle. Epinephrine and cAMP enhance the formation of ketopentoses and sedoheptulose with glycogen as a main carbohydrate source, which is most pronounced in the experiments with cold preincubation. The phosphorylase system mediate influence of epinephrine and cAMP on the nonoxidative reactions products may be assumed.

  11. [Effect of prostaglandins F2 and F2 alpha on the pentosephosate pathway in human blood platelets].

    PubMed

    Makarov, S A; Kudriavtseva, G V; Kolotilova, A I

    1983-01-01

    Rates of glucose-6-phosphate oxidation and formation of sedoheptulose-7-phosphate were increased after 10 min preincubation of human blood platelets with prostaglandins F2 and F2a. When the preincubation was prolonged up to 60 min, the prostaglandins activating effect was manifested only as an increase in sedoheptulose-7-phosphate content. Preincubation of thrombocytes with dibutyryl-cAMP led to an increase in the rate of ribose-5-phosphate consumption as well as of sedoheptulose-7-phosphate formation. Chlorpromazine hydrochloride decreased the rates of glucose-6-phosphate and 6-phosphogluconate oxidation in extracts of human thrombocytes and inhibited the activating effect of prostaglandins on sedoheptulose-7-phosphate formation.

  12. Pentose fermentation by recombinant zymomonas

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  13. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

    1998-03-10

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  14. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  15. Pentose fermentation by recombinant Zymomonas

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

    1998-01-27

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  16. Single zymomonas mobilis strain for xylose and arabinose fermentation

    DOEpatents

    Zhang, Min; Chou, Yat-Chen; Picataggio, Stephen K.; Finkelstein, Mark

    1998-01-01

    This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol.

  17. 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

  18. Comparative Studies on Biochemical Properties of Protein Synthesis of an Archae-Bacteria Thermoplasma-Sp

    NASA Astrophysics Data System (ADS)

    Ohba, Masayuki; Oshima, Tairo

    1982-12-01

    An acido-thermophillic archaebacteria,Thermoplasma strain KO-2, produced poly(A) containing RNA. The isolated poly(A)RNA showed the messenger activity in a cell-free extract of rabbit reticulocyte, indicating that the RNA is mRNA of the archaebacteria. 7-Methylgluanosine 5'-phosphate did not inhibit the reaction, suggesting that the cap structure is not present in the messenger. These results may suggest that poly(A) containing messenger arised at very early stage of evolution prior to the divergence between archaebacteria and eukaryotes.

  19. MicroCommentary: A New Role for Coenzyme F420 in Aflatoxin Reduction by Soil Mycobacteria

    SciTech Connect

    Graham, David E

    2010-01-01

    Hepatotoxic aflatoxins have found a worthy adversary in two new families of bacterial oxidoreductases. These enzymes use the reduced coenzyme F420 to initiate the degradation of furanocoumarin compounds, including the major mycotoxin products of Aspergillus flavus. Along with pyridoxalamine 5 -phosphate oxidases and aryl nitroreductases, these proteins form a large and versatile superfamily of flavin and deazaflavin-dependent oxidoreductases. F420-dependent members of this family appear to share a common mechanism of hydride transfer from the reduced deazaflavin to the electron-deficient ring systems of their substrates.

  20. Single Zymomonas mobilis strain for xylose and arabinose fermentation

    DOEpatents

    Zhang, M.; Chou, Y.C.; Picataggio, S.K.; Finkelstein, M.

    1998-12-01

    This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol. 6 figs.

  1. Cloning, purification, crystallization and preliminary crystallographic analysis of a ribokinase from Staphylococcus aureus

    PubMed Central

    Wang, Lin; Wang, Haipeng; Ruan, Jianbin; Tian, Changlin; Sun, Baolin; Zang, Jianye

    2009-01-01

    The gene SA239 from Staphylococcus aureus encodes a ribokinase that catalyzes the phosphorylation of d-ribose to produce ribose-5-phosphate. Sa239 was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.9 Å resolution and belonged to space group P6122 or P6522, with unit-cell parameters a = b = 91.8, c = 160.7 Å. Preliminary crystallographic analysis revealed that the Matthews coefficient V M was 3.01 Å3 Da−1, indicating the presence of one molecule in the asymmetric unit. PMID:19478434

  2. Electrochemistry of N-n-undecyl-N'-(sodium-p-aminobenzenesulfonate) thiourea and its interaction with bovine serum albumin.

    PubMed

    Luo, Hongxia; Du, Yang; Guo, Zhi-Xin

    2009-02-01

    In pH 5.5 phosphate buffer solution, N-n-undecyl-N'-(sodium-p-aminobenzenesulfonate) thiourea (UPT) produced a pair of redox peaks on the bare glassy carbon electrode. At the multi-walled carbon nanotube (MWNT) modified electrode, the electrochemical behavior of UPT enhanced greatly. In the presence of bovine serum albumin (BSA), the peak currents of UPT decreased linearly due to the formation of a super-molecular complex. This method was successfully applied to the determination of BSA in a bovine serum sample.

  3. Formation of nucleoside 5'-polyphosphates under potentially prebiological conditions

    NASA Technical Reports Server (NTRS)

    Lohrmann, R.

    1976-01-01

    The characteristics and efficiencies of biochemical reactions involving nucleoside 5'-diphosphates and -triphosphates (important substrates of RNA and DNA synthesis) under conditions corresponding to the primitive prebiotic earth are investigated. Urea catalysis of the formation of linear inorganic polyphosphates and metal ions promoting the reactions are discussed. Linear polyphosphate was incubated with Mg(++) in the presence of a nucleoside 5'-phosphate, to yield nucleoside 5'-polyphosphates when products are dried, while Mg(++) prompts depolymerization to trimetaphosphate in aqueous solutions. Plausible biogenetic pathways are examined.

  4. The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

    PubMed

    Junowicz, E; Charm, S E

    1976-03-25

    The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography. PMID:1260016

  5. Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family

    PubMed Central

    2016-01-01

    The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3′,4,4′,5,5′-hexakisphosphate [BiPh(3,3′,4,4′,5,5′)P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3′,4,4′,5,5′)P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3′,4,4′,5,5′)P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B–BiPh(3,3′,4,4′,5,5′)P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed “moving metal” mechanism

  6. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  7. Synthesis of arabinitol 1-phosphate and its use for characterization of arabinitol-phosphate dehydrogenase.

    PubMed

    Soroka, Nikolai V; Kulminskaya, Anna A; Eneyskaya, Elena V; Shabalin, Konstantin A; Uffimtcev, Andrei V; Povelainen, Mira; Miasnikov, Andrei N; Neustroev, Kirill N

    2005-03-21

    D-arabinitol 1-phosphate (Ara-ol1-P), a substrate for D-arabinitol-phosphate dehydrogenase (APDH), was chemically synthesized from D-arabinonic acid in five steps (O-acetylation, chlorination, reduction, phosphorylation, and de-O-acetylation). Ara-ol1-P was used as a substrate for the characterization of APDH from Bacillus halodurans. APDH converts Ara-ol1-P to xylulose 5-phosphate in the oxidative reaction; both NAD(+) and NADP(+) were accepted as co-factors. Kinetic parameters for the oxidative and reductive reactions are consistent with a ternary complex mechanism.

  8. An aqueous friendly chemosensor derived from vitamin B6 cofactor for colorimetric sensing of Cu2 + and fluorescent turn-off sensing of Fe3 +

    NASA Astrophysics Data System (ADS)

    Sharma, Darshna; Kuba, Aman; Thomas, Rini; Kumar, Rajender; Choi, Heung-Jin; Sahoo, Suban K.

    2016-01-01

    Chemosensor L derived from vitamin B6 cofactor pyridoxal-5-phosphate was investigated for the selective detection of Cu2 + and Fe3 + in aqueous medium. Sensor L formed a 1:1 complex with Cu2 + and displays a perceptible color change from colorless to yellow brown with the appearance of a new charge transfer band at ~ 450 nm. In contrast, the fluorescence of L was quenched selectively in the presence of Fe3 + without any interference from other metal ions including Cu2 +.

  9. Evidence for the presence of phosphoriboisomerase and ribulose-1,5-diphosphate carboxylase in extracts of Desulfovibrio vulgaris.

    PubMed

    Alvarez, M; Barton, L L

    1977-07-01

    Cell extracts of Desulfovibrio vulgaris were found to incorporate 14CO2 into acid-stable products when ribose-5-phosphate or ribulose-1,5-diphosphate was used as a substrate. This CO2 fixation required adenosine triphosphate and produced 3-phosphoglyceric acid as one of the products. The assimilation of CO2 by pentose phosphates was unrelated to the pyruvate-CO2 exchange reaction. The pyruvate-CO2 exchange did not require adenosine triphosphate, did not produce phosphorylated compounds, and, unlike the pentose phosphate system, required an acidic protein fraction for activity.

  10. Biosynthesis of D-arabinose in mycobacteria - a novel bacterial pathway with implications for antimycobacterial therapy.

    PubMed

    Wolucka, Beata A

    2008-06-01

    Decaprenyl-phospho-arabinose (beta-D-arabinofuranosyl-1-O-monophosphodecaprenol), the only known donor of d-arabinose in bacteria, and its precursor, decaprenyl-phospho-ribose (beta-D-ribofuranosyl-1-O-monophosphodecaprenol), were first described in 1992. En route to D-arabinofuranose, the decaprenyl-phospho-ribose 2'-epimerase converts decaprenyl-phospho-ribose to decaprenyl-phospho-arabinose, which is a substrate for arabinosyltransferases in the synthesis of the cell-wall arabinogalactan and lipoarabinomannan polysaccharides of mycobacteria. The first step of the proposed decaprenyl-phospho-arabinose biosynthesis pathway in Mycobacterium tuberculosis and related actinobacteria is the formation of D-ribose 5-phosphate from sedoheptulose 7-phosphate, catalysed by the Rv1449 transketolase, and/or the isomerization of d-ribulose 5-phosphate, catalysed by the Rv2465 d-ribose 5-phosphate isomerase. d-Ribose 5-phosphate is a substrate for the Rv1017 phosphoribosyl pyrophosphate synthetase which forms 5-phosphoribosyl 1-pyrophosphate (PRPP). The activated 5-phosphoribofuranosyl residue of PRPP is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenyl phosphate, thus forming 5'-phosphoribosyl-monophospho-decaprenol. The dephosphorylation of 5'-phosphoribosyl-monophospho-decaprenol to decaprenyl-phospho-ribose by the putative Rv3807 phospholipid phosphatase is the committed step of the pathway. A subsequent 2'-epimerization of decaprenyl-phospho-ribose by the heteromeric Rv3790/Rv3791 2'-epimerase leads to the formation of the decaprenyl-phospho-arabinose precursor for the synthesis of the cell-wall arabinans in Actinomycetales. The mycobacterial 2'-epimerase Rv3790 subunit is similar to the fungal D-arabinono-1,4-lactone oxidase, the last enzyme in the biosynthesis of D-erythroascorbic acid, thus pointing to an evolutionary link between the D-arabinofuranose- and L-ascorbic acid-related pathways. Decaprenyl-phospho-arabinose has been a lead compound for the

  11. Cyclic aristeromycin diphosphate ribose: a potent and poorly hydrolysable Ca(2+)-mobilising mimic of cyclic adenosine diphosphate ribose.

    PubMed

    Bailey, V C; Fortt, S M; Summerhill, R J; Galione, A; Potter, B V

    1996-02-01

    Cyclic aristeromycin diphosphate ribose, a carbocyclic analogue of cyclic adenosine diphosphate ribose, was synthesised using a chemo-enzymatic route involving activation of aristeromycin 5'-phosphate by diphenyl phosphochloridate. The calcium-releasing properties of this novel analogue were investigated in sea urchin egg homogenates. While cyclic aristeromycin diphosphate ribose has a calcium release profile similar to that of cyclic adenosine diphosphate ribose (EC50 values are 80 nM and 30 nM, respectively), it is degraded significantly more slowly (t1/2 values are 170 min and 15 min, respectively) and may, therefore, be a useful tool to investigate the activities of cyclic adenosine diphosphate ribose. PMID:8603694

  12. Disease activity and severity in early inflammatory arthritis predict hand cortical bone loss

    PubMed Central

    Pye, Stephen R.; Adams, Judith E.; Ward, Kate A.; Bunn, Diane K.; Symmons, Deborah P. M.

    2010-01-01

    Objectives. To determine the influence of disease-related variables on hand cortical bone loss in women with early inflammatory arthritis (IA), and whether hand cortical bone mass predicts subsequent joint damage. Method. Adults aged ≥16 years with recent onset of IA were recruited to the Norfolk Arthritis Register between 1990 and 1998, and followed prospectively. At baseline, patients had their joints examined for swelling and tenderness and had CRP and disease activity 28-joint assessment score (DAS-28) measured. Radiographs of the hands were performed in a subgroup of patients at Year 1 and at follow-up, which were assessed using digital X-ray radiogrammetry (DXR). They were also evaluated for the presence of erosions using Larsen’s method. Linear mixed models were used to investigate whether disease-related factors predicted change in DXR–areal bone mineral density (BMDa). We also evaluated whether DXR–BMDa predicted the subsequent occurrence of erosive disease. Results. Two hundred and four women, mean (s.d.) age 55.1 (14.0) years, were included. Median follow-up between radiographs was 4 years. The mean within-subject change in BMDa was 0.024 g/cm2 equivalent to 1% decline per year. After adjustment for age, height and weight, compared with those within the lower tertile for CRP, those in the upper tertile had greater subsequent loss of bone. This was true also for DAS-28 and Larsen score. Among those without erosions on the initial radiograph (121), DXR–BMDa at baseline did not predict the new occurrence of erosions. Conclusion. Increased disease activity and severity are associated with accelerated bone loss. However, lower BMDa did not predict the new occurrence of erosive disease. PMID:20573690

  13. Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics

    SciTech Connect

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. S.; Ahring, Birgitte K.; Linggi, Bryan E.

    2013-06-19

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. We found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.

  14. Do medical students’ scores using different assessment instruments predict their scores in clinical reasoning using a computer-based simulation?

    PubMed Central

    Fida, Mariam; Kassab, Salah Eldin

    2015-01-01

    Purpose The development of clinical problem-solving skills evolves over time and requires structured training and background knowledge. Computer-based case simulations (CCS) have been used for teaching and assessment of clinical reasoning skills. However, previous studies examining the psychometric properties of CCS as an assessment tool have been controversial. Furthermore, studies reporting the integration of CCS into problem-based medical curricula have been limited. Methods This study examined the psychometric properties of using CCS software (DxR Clinician) for assessment of medical students (n=130) studying in a problem-based, integrated multisystem module (Unit IX) during the academic year 2011–2012. Internal consistency reliability of CCS scores was calculated using Cronbach’s alpha statistics. The relationships between students’ scores in CCS components (clinical reasoning, diagnostic performance, and patient management) and their scores in other examination tools at the end of the unit including multiple-choice questions, short-answer questions, objective structured clinical examination (OSCE), and real patient encounters were analyzed using stepwise hierarchical linear regression. Results Internal consistency reliability of CCS scores was high (α=0.862). Inter-item correlations between students’ scores in different CCS components and their scores in CCS and other test items were statistically significant. Regression analysis indicated that OSCE scores predicted 32.7% and 35.1% of the variance in clinical reasoning and patient management scores, respectively (P<0.01). Multiple-choice question scores, however, predicted only 15.4% of the variance in diagnostic performance scores (P<0.01), while students’ scores in real patient encounters did not predict any of the CCS scores. Conclusion Students’ scores in OSCE are the most important predictors of their scores in clinical reasoning and patient management using CCS. However, real patient

  15. Land Snails as a Diet Diversification Proxy during the Early Upper Palaeolithic in Europe

    PubMed Central

    Fernández-López de Pablo, Javier; Badal, Ernestina; Ferrer García, Carlos; Martínez-Ortí, Alberto; Sanchis Serra, Alfred

    2014-01-01

    Despite the ubiquity of terrestrial gastropods in the Late Pleistocene and Holocene archaeological record, it is still unknown when and how this type of invertebrate resource was incorporated into human diets. In this paper, we report the oldest evidence of land snail exploitation as a food resource in Europe dated to 31.3-26.9 ka yr cal BP from the recently discovered site of Cova de la Barriada (eastern Iberian Peninsula). Mono-specific accumulations of large Iberus alonensis land snails (Ferussac 1821) were found in three different archaeological levels in association with combustion structures, along with lithic and faunal assemblages. Using a new analytical protocol based on taphonomic, microX-Ray Diffractometer (DXR) and biometric analyses, we investigated the patterns of selection, consumption and accumulation of land snails at the site. The results display a strong mono-specific gathering of adult individuals, most of them older than 55 weeks, which were roasted in ambers of pine and juniper under 375°C. This case study uncovers new patterns of invertebrate exploitation during the Gravettian in southwestern Europe without known precedents in the Middle Palaeolithic nor the Aurignacian. In the Mediterranean context, such an early occurrence contrasts with the neighbouring areas of Morocco, France, Italy and the Balkans, where the systematic nutritional use of land snails appears approximately 10,000 years later during the Iberomaurisian and the Late Epigravettian. The appearance of this new subsistence activity in the eastern and southern regions of Spain was coeval to other demographically driven transformations in the archaeological record, suggesting different chronological patterns of resource intensification and diet broadening along the Upper Palaeolithic in the Mediterranean basin. PMID:25141047

  16. Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana.

    PubMed

    Yang, Yongheng; Huang, Suzhen; Han, Yulin; Yuan, Haiyan; Gu, Chunsun; Wang, Zhongwei

    2015-01-01

    Plant growth and secondary metabolism are commonly regulated by external cues such as light, temperature and water availability. In this study, the influences of low and high temperatures, dehydration, photoperiods, and different growing stages on the changes of steviol glycosides (SGs) contents and transcription levels of fifteen genes involved in SGs biosynthesis of Stevia rebaudiana Bertoni were examined using HPLC and RT-PCR. The observations showed that the transcript levels of all the fifteen genes were maximum under 25 °C treatment, and the transcription of SrDXS, SrDXR, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI, SrGGDPS, SrCPPS1, SrUGT85C2 and SrUGT76G1 were restrained both in low temperature (15 °C) and high temperature (35 °C). Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration. To elucidate the effect of photoperiods, the plants were treated by different simulated photoperiods (8 L/16 D, 1 0L/14 D, 14 L/10 D and 16 L/8 D), but no significant transcription changes were observed. In the study of growing stages, there were evident changes of SGs contents, and the transcript levels of all the fifteen genes were minimal in fast growing period, and exhibited evident increase both in flower-bud appearing stage and flowering stage. The obtained results strongly suggest that the effect of environmental cues on steviol glycosides contents and transcription of corresponding biosynthetic genes in S. rebaudiana is significant. It is worth to study deeply.

  17. Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics.

    PubMed

    Hess, Becky M; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C; Wiley, H Steven; Ahring, Birgitte K; Linggi, Bryan

    2013-01-01

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions. PMID:23840410

  18. Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics

    PubMed Central

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. Steven; Ahring, Birgitte K.; Linggi, Bryan

    2013-01-01

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions. PMID:23840410

  19. The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana.

    PubMed

    Caparrós-Martín, José A; McCarthy-Suárez, Iva; Culiáñez-Macià, Francisco A

    2014-09-01

    The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943-954, 2013). In the presence of Mg(2+) ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on D-ribose-5-phosphate, 2-deoxy-D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-fructose-1-phosphate, D-glucose-6-phosphate, DL-glycerol-3-phosphate, and D-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous DL-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for DL-glycerol-3-phosphate. PMID:24915748

  20. PLP and PMP radicals: a new paradigm in coenzyme B6 chemistry.

    PubMed

    Agnihotri, G; Liu, H W

    2001-08-01

    Enzymes frequently rely on a broad repertoire of cofactors to perform chemically challenging transformations. The B6 coenzymes, composed of pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), are used by many transaminases, racemases, decarboxylases, and enzymes catalyzing alpha,beta and beta,gamma-eliminations. Despite the variety of reactions catalyzed by B6-dependent enzymes, the mechanism of almost all such enzymes is based on their ability to stabilize high-energy anionic intermediates in their reaction pathways by the pyridinium moiety of PLP/PMP. However, there are two notable exceptions to this model, which are discussed in this article. The first enzyme, lysine 2,3-aminomutase, is a PLP-dependent enzyme that catalyzes the interconversion of L-lysine to L-beta-lysine using a one-electron-based mechanism utilizing a [4Fe-4S] cluster and S-adenosylmethionine. The second enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase, is a PMP-dependent enzyme involved in the formation of 3,6-dideoxysugars in bacteria. This enzyme also contains an iron-sulfur cluster and uses a one-electron based mechanism to catalyze removal of a C-3 hydroxy group from a 4-hexulose. In both cases, the participation of free radicals in the reaction pathway has been established, placing these two B6-dependent enzymes in an exclusive class by themselves.

  1. A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima.

    PubMed

    Moustafa, Hanan M A; Zaghloul, Taha I; Zhang, Y-H Percival

    2016-05-15

    Phosphopentomutase (PPM) catalyzes the interconversion of α-D-(deoxy)-ribose 1-phosphate and α-D-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside phosphorylase for determining PPM activities on D-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl2 (0.1 mM) and 50 μM α-D-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of Km and kcat are 1.2 mM and 185 s(-1) at 90 °C, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date. PMID:26924489

  2. Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus.

    PubMed

    Aihara, Takayuki; Ito, Toshiya; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Sekine, Masae; Homma, Hiroshi; Yohda, Masafumi

    2016-07-01

    Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus. PMID:27094682

  3. Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera.

    PubMed

    Sanchita; Singh, Swati; Sharma, Ashok

    2014-11-01

    Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-D-xylulose-5-phosphate synthase, 1-Deoxy-D-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.

  4. Purification of low-concentration phenazine-1-carboxylic acid from fermentation broth of Pseudomonas sp. M18 via free flow electrophoresis with gratis gravity.

    PubMed

    Shao, Jing; Fan, Liu-Yin; Zhang, Wei; Guo, Chen-Gang; Li, Si; Xu, Yu-Quan; Cao, Cheng-Xi

    2010-10-01

    The low-concentration phenazine-1-carboxylic acid (PCA) ( = 0.3 mM) extracted from fermentation broth of Pseudomonas sp. M18 was selected to be purified with a newly facile free flow electrophoresis (FFE) device with gratis gravity. Three factors of pH value and concentration of background buffer, and the cooling circle of FFE device were investigated for the purification of PCA in the FFE device. It was found that the pH value and concentration of background buffer had mild influences on the separation of PCA whether with cooling circle or not. However, the cooling circle had a much greater impact on the separation of PCA. The controlling of the band zone of PCA in FFE chamber would be difficult if without cooling circle, while the controlling would become easy if with cooling circle. Under the optimal conditions (10 mM pH 5.5 phosphate as background buffer, 30 mM pH 5.5 phosphate buffer as electrode solution, 5.46 mL/min background flux, 10 min residence time of injected sample, and 500 V), PCA could be continuously prepared from its impurities with relative high purity. The flux of sample injection was 115 μL/min, viz. 7 mL sample throughput per hour, and the recovery was up to 85%. All of the experiments indicated that the FFE technique was a good alternative tool for the study on natural biological control agents.

  5. Difference in the xylitol sensitivity of acid production among Streptococcus mutans strains and the biochemical mechanism.

    PubMed

    Miyasawa-Hori, H; Aizawa, S; Takahashi, N

    2006-08-01

    Xylitol inhibits the glycolysis and growth of Streptococcus mutans, but to different degrees among strains. Thus, we studied the biochemical mechanism through which the inhibition varies, using S. mutans strains ATCC 31989, NCTN 10449, and NCIB 11723, which are highly sensitive, moderately sensitive, and resistant to xylitol, respectively, under strictly anaerobic conditions such as those found in deep layers of dental plaque. Xylitol (30 mM) decreased the rate of acid production from glucose (10 mM) in ATCC 31989, NCTC 10449, and NCIB 11723 by 86, 26, and 0%, respectively. The activities of the xylitol : phosphoenolpyruvate phosphotransferase system (PEP-PTS) relative to those of glucose : PEP-PTS were 120, 16, and 3%, respectively. In ATCC 31989 and NCTC 10449, intracellular accumulation of xylitol 5-phosphate and decreases of fructose 1,6-bisphosphate and glucose 6-phosphate were observed. Furthermore, in the presence of xylitol (30 mM), glucose : PEP-PTS activities decreased by 34, 17, and 0%, respectively. These findings indicated that the higher the xylitol : PEP-PTS activity was and the more effectively xylitol decreased glucose : PEP-PTS activity, the more sensitive the strain was to xylitol. These results suggest that the following inhibitory mechanisms are active in the xylitol-sensitive mutans streptococci: direct inhibition of glycolytic enzymes by xylitol 5-phosphate derived from xylitol : PEP-PTS and, possibly, indirect inhibition through competition for the phosphoryl donor, HPr-P, between glucose and xylitol : PEP-PTSs.

  6. Positive correlation between PSI response and oxidative pentose phosphate pathway activity during salt stress in an intertidal macroalga.

    PubMed

    Huan, Li; Xie, Xiujun; Zheng, Zhenbing; Sun, Feifei; Wu, Songcui; Li, Moyang; Gao, Shan; Gu, Wenhui; Wang, Guangce

    2014-08-01

    Studies have demonstrated that photosynthetic limitations and starch degradation are responses to stress; however, the relationship between the two is seldom described in detail. In this article, the effects of salt stress on photosynthesis, the levels of NADPH and total RNA, the starch content and the activities of glucose-6-phosphate dehydrogenase (G6PDH) and ribulose-5-phosphate kinase (RPK) were evaluated. In thalli that underwent salt treatments, the cyclic electron flow through PSI showed greater stress tolerance than the flow through PSII. Even though the linear electron flow was suppressed by DCMU, the cyclic electron flow still operated. The electron transport rate I (ETRI) increased as the salinity increased when the thalli recovered in seawater containing DCMU. These results suggested that PSI receives electrons from a source other than PSII. Furthermore, the starch content and RPK activity decreased, while the content of NADPH and total RNA, and the activity of G6PDH increased under salt stress. Soluble sugar from starch degradation may enter the oxidative pentose phosphate pathway (OPPP) to produce NADPH and ribose 5-phosphate. Data analysis suggests that NADPH provides electrons for PSI in Ulva prolifera during salt stress, the OPPP participates in the stress response and total RNA is synthesized in excess to assist recovery.

  7. The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana.

    PubMed

    Caparrós-Martín, José A; McCarthy-Suárez, Iva; Culiáñez-Macià, Francisco A

    2014-09-01

    The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943-954, 2013). In the presence of Mg(2+) ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on D-ribose-5-phosphate, 2-deoxy-D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-fructose-1-phosphate, D-glucose-6-phosphate, DL-glycerol-3-phosphate, and D-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous DL-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for DL-glycerol-3-phosphate.

  8. 5'-C-Malonyl RNA: Small Interfering RNAs Modified with 5'-Monophosphate Bioisostere Demonstrate Gene Silencing Activity.

    PubMed

    Zlatev, Ivan; Foster, Donald J; Liu, Jingxuan; Charisse, Klaus; Brigham, Benjamin; Parmar, Rubina G; Jadhav, Vasant; Maier, Martin A; Rajeev, Kallanthottathil G; Egli, Martin; Manoharan, Muthiah

    2016-04-15

    5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.

  9. Stability of hydrophilic vitamins mixtures in the presence of electrolytes and trace elements for parenteral nutrition: a nuclear magnetic resonance spectroscopy investigation.

    PubMed

    Uccello-Barretta, Gloria; Balzano, Federica; Aiello, Federica; Falugiani, Niccolò; Desideri, Ielizza

    2015-03-25

    In total parenteral nutrition (TPN), especially in the case of preterm infants, simultaneous administration of vitamins and trace elements is still a problematic issue: guidelines put in evidence the lack of specific documentation. In this work NMR spectroscopy was applied to the study of vitamins (pyridoxine hydrochloride, thiamine nitrate, riboflavin-5'-phosphate and nicotinamide) stability in presence of salts and trace elements. Vitamins in D2O were first analyzed by (1)H NMR spectroscopy in absence of salts and trace elements; changes in chemical shifts or in diffusion coefficients, measured by NMR DOSY technique, were analyzed. The effects of salts and trace elements on single vitamins and on their admixtures were then investigated by performing quantitative analyses during 48h. Selected vitamins are subject to intermolecular interactions. No degradative effects were observed in presence of salts and trace elements. Only riboflavin-5'-phosphate is subject to precipitation in presence of divalent cations; however, at low concentration and in presence of other vitamins this effect was not observed. Solutions analyzed, in the condition of this study, are stable for at least 48h and vitamins and trace elements can be administered together in TPN.

  10. General Properties, Occurrence, and Preparation of Carbohydrates

    NASA Astrophysics Data System (ADS)

    Robyt, John F.

    D-Glucose and its derivatives and analogues, N-acetyl-D-glucosamine, N-acetyl-D-muramic acid, D-glucopyranosyl uronic acid, and D-glucitol represent 99.9% of the carbohydrates on the earth. D-Glucose is found in the free state in human blood and in the combined state in disaccharides, sucrose, lactose, and α,α-trehalose, in cyclic dextrins, and in polysaccharides, starch, glycogen, cellulose, dextrans; N-acetyl-D-glucosamine and an analogue N-acetyl-D-muramic acid are found in bacterial cell wall polysaccharide, murein, along with teichoic acids made up of poly-glycerol or -ribitol phosphodiesters. Other carbohydrates, D-mannose, D-mannuronic acid, D-galactose, N-acetyl-D-galactosamine, D-galacturonic acid, D-iduronic acid, L-guluronic acid, L-rhamnose, L-fucose, D-xylose, and N-acetyl-D-neuraminic acid are found in glycoproteins, hemicelluloses, glycosaminoglycans, and polysaccharides of plant exudates, bacterial capsules, alginates, and heparin. D-Ribofuranose-5-phosphate is found in many coenzymes and is the backbone of RNAs (ribonucleic acid), and 2-deoxy-D-ribofuranose-5-phosphate is the backbone of DNA (deoxyribonucleic acid). D-Fructofuranose is found in sucrose, inulin, and levan. The general properties and occurrence of these carbohydrates and general methods of isolation and preparation of carbohydrates are presented.

  11. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

  12. D-ribose competitively reverses inhibition by D-psicose of larval growth in Caenorhabditis elegans.

    PubMed

    Sato, Masashi; Yokoi, Nobutoshi; Kurose, Hiroyuki; Yamasaki, Toru

    2009-05-01

    D-Psicose inhibits the growth of L1 stage Caenorhabditis elegans. Sugars, involved in the pentose phosphate pathway, were examined for their ability to reverse the inhibition. Among these sugars, D-ribose specifically exerted reversing activity in a competitive manner. The ingested sugars are probably phosphorylated, although it remains to be seen whether D-psicose is phosphorylated. The structural similarity of D-psicofuranose 6-phosphate (Pf6P) or D-psicofuranose (Pf) to D-ribofuranose 5-phosphate (Rf5P) suggests that Pf6P or Pf is reversibly docked in the active site(s) of ribose-5-phosphate isomerase(s) to act as an antimetabolite to Rf5P, leading to inhibition of the biosynthesis of nucleic acids. D-Psicose was much less potent against the L4 stage than against the L1 stage. This is probably because in the L4 stage the somatic cell lineages come to an end and the number of germ-line nuclei increases to about 1000.

  13. The greenhouse effect: Physiological changes in plants

    SciTech Connect

    Beard, R.; Harrison, M. )

    1990-05-01

    Elevated atmospheric carbon dioxide is timely topic of study for all biology students at all levels. The stimulatory effect of elevated atmospheric carbon dioxide (700 {mu}l/l) on plant growth, flower induction, protein production and the Calvin-Benson cycle can be easily demonstrated in seedlings in student laboratories. In our lab, the shoot growth of rapid cycling brassicas (Wisconsin fast plants) was measured under ambient and elevated CO{sub 2} conditions for three weeks. Plants grown under elevated CO{sub 2} conditions were significantly taller and showed earlier flower development. These plants also contained greater protein content per gram fresh weight. Crude leaf extracts was used as a source of pentose-5-isomerase which controls the conversion of ribose-5-phosphate to ribulose-5-phosphate in carbon fixation. The activity of this enzyme was measured spectrophotometrically and found to be somewhat greater in plants grown under the higher CO{sub 2} conditions. These physiological changes associated with elevated CO{sub 2} can be used as an introduction to the greenhouse effect as well as to study the regulation of carbon fixation.

  14. Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli.

    PubMed

    De Muynck, Cassandra; Van der Borght, Jef; De Mey, Marjan; De Maeseneire, Sofie L; Van Bogaert, Inge N A; Beauprez, Joeri; Soetaert, Wim; Vandamme, Erick

    2007-10-01

    L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.

  15. Ribulose diphosphate carboxylase/oxygenase. IV. Regulation by phosphate esters.

    PubMed

    Ryan, F J; Tolbert, N E

    1975-06-10

    The stimulation or inhibition of ribulose diphosphate oxygenase by a variety of compounds is compared with the reported effects on these compounds on the ribulose diphosphate carboxylase activity. A possible transition state analog of ribulose diphosphate, 2-carboxyribitol 1, 5-diphosphate, at a molar ratio of inhibitor to enzyme of 10 to 1, irreversibly inactivates the oxygenase and carboxylase activities. This is consistent with the hypothesis that there may be a single active site for both the carboxylase and oxygenase activities. Several compounds of the reductive pentose photosynthetic carbon cycle act as effectors of the ribulose diphosphate oxygenase in a manner complementary to their reported effect upon the carboxylase. Ribose 5-phosphate inhibits the oxygenase with an apparent Ki of 1.8 mM, but it is reported to activate the carboxylase; fructose 6-phosphate and glucose 6-phosphate act similarly but are less effective than ribose 5-phosphate. Fructose 1. 6-diphosphate stimulates the oxygenase at low magnesium ion concentrations. The stimulatory effect of 6-phosphogluconate on the oxygenase is associated with a 3-fold reduction of the Km (Mg2+). ATP inhibits the oxygenase but has been reported to stimulate the carboxylase; pyrophosphate acts in an opposite manner. From these results it appears that the ratio of carboxylase to oxygenase activity may be a variable factor with predictable subsequent alteration in the ratio between photosynthetic CO2 fixation and photorespiration.

  16. Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase

    SciTech Connect

    Han,Q.; Gao, Y.; Robinson, H.; Li, J.

    2008-01-01

    Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant a-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate {beta}-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and {alpha}7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

  17. Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds.

    PubMed

    Ghosh, B; De, B P; Biswas, B B

    1984-01-01

    A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of Mr 32,000 each was discernible. Km values for NAD+ and myo-inositol 1-phosphate have been recorded as 2.8 X 10(-4) and 5.0 X 10(-4) M, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD+ are reduced.

  18. Nonenzymatic, template-directed ligation of oligoribonucleotides is highly regioselective for the formation of 3'-5' phosphodiester bonds

    NASA Technical Reports Server (NTRS)

    Rohatgi, R.; Bartel, D. P.; Szostak, J. W.

    1996-01-01

    We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'-5' rather than 2'-5' phosphodiester bonds. Formation of the 3'-5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'-5'- to 2'-5'-linked products was 10-15:1 when the 5'-phosphate was activated as the imidazolide, and 60-80:1 when the 5'-phosphate was activated by the formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'-5'-linkages in oligonucleotide ligation is primarily due to occurence of ligation within the context of an extended Watston-Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.

  19. Coordinateendonucleolytic 5' and 3' trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

    SciTech Connect

    Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer; F., Lawrence

    2008-02-18

    Previous work showed that, in the presence of DNA-PK, Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5' {yields} 3' exonucleolytic resection of double-stranded DNA. This resection required a 5'-phosphate but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent, and did not require a 5'-phosphate. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nucleotides from the 5'-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.

  20. Interaction of monosaccharides and related compounds with oxocations of Mo(VI), W(VI) and U(VI) studied by NMR spectroscopy

    SciTech Connect

    Geraldes, C.F.G.C.; Castro, M.M.C.A.; Saraiva, M.E.; Aureliano, M.; Dias, B.A.

    1988-05-01

    Proton, /sup 13/C and /sup 31/P nuclear magnetic resonance spectroscopy has been used to study the complexation of Mo(VI), W(VI) and U(VI) oxocations with various aldoses, cyclic polyols and ribose-5-phosphate in aqueous solution. The aldoses D-mannose, D-lyxose and D-ribose form tridentate complexes with Mo(VI) and W(VI) at pH similarly ordered 5, via the 1,2,3-hydroxyl groups, which are cis to each other in these sugars. Other aldoses, like D-arabinose, D-glucose, D-xylose and D-galactose form weaker bidentate complexes with those ions because they can only use the 1 and 3-cis hydroxyl groups in metal binding. These bidentate interactions also take place in the binding of U(VI) to D-mannose and D-ribose, at pH similarly ordered 10. However, sugars having 1,3,5-hydroxyl groups in the cis position do not form stable chelates with these oxocations, possibly due to steric crowding. In the case of ribose-5-phosphate, the phosphate group is the exclusive binding site for the three oxocations, except for U(VI) at very basic pH (pH > 10), where the hydroxyl groups also interact with UO/sub 2//sup 2 +/.

  1. A history of the isolation and identification of vitamin B(6).

    PubMed

    Rosenberg, Irwin H

    2012-01-01

    In the 1930s, Rudolf Peters showed that young rats kept on a semi-synthetic diet with added thiamin and riboflavin but no other supplement developed 'rat acrodynia', a condition characterized by severe cutaneous lesions. In 1934, Paul György showed that the factor which cured 'rat acrodynia' was vitamin B(6). Other studies soon showed that vitamin B(6) deficiency produced convulsions in rats, pigs, and dogs, and a microcytic anemia in certain animals. Samuel Lepkovsky isolated and crystallized vitamin B(6) in 1938. The following year, Leslie Harris and Karl Folkers, and Richard Kuhn and his associates independently showed that vitamin B(6) was a pyridine derivative, 3-hydroxy-4,5-dihydroxy-methyl-2-methyl-pyridine. György proposed the term pyridoxine for this derivative. Esmond Snell developed a microbiological growth assay in 1942 that led to the characterization of pyridoxamine, the animated product of pyridoxine, and pyridoxal, the formyl derivative of pyridoxine. Further studies showed that pyridoxal, pyridoxamine, and pyridoxine have largely equal activity in animals and owe their vitamin activity to the ability of the organism to convert them into the enzymatically active form pyridoxal-5-phosphate. Pyridoxal-5-phosphate plays a role in a wide variety of enzyme systems, especially in the metabolic utilization and transformation of amino acids.

  2. The metal-binding sites of glycose phosphates.

    PubMed

    Gilg, Kathrin; Mayer, Tobias; Ghaschghaie, Natascha; Klüfers, Peter

    2009-10-14

    In aqueous solution, the reducing sugar phosphates D-arabinose 5-phosphate, D-ribose 5-phosphate, D-fructose 1,6-bisphosphate, D-fructose 6-phosphate, D-glucose 6-phosphate and D-mannose 6-phosphate provide metal-binding sites at their glycose core on reaction with Pd(II)(en) or M(III)(tacn) residues (M = Ga, Co; en = ethylenediamine, tacn = 1,4,7-triazacyclononane). The individual species were detected by one- and two-dimensional NMR spectroscopy. The coordination patterns are related to the metal-binding modes of the respective parent glycoses. In detail, ribo- and arabinofuranose phosphate favour kappaO(1,3) coordination, whereas the ketofuranose core of fructose phosphate and fructose bisphosphate provides the kappaO(2,3) chelator thus maintaining the configuration of the respective major solution anomer. On palladium excess, D-fructose 6-phosphate is metallated twice in a unique kappaO(1,3):kappaO(2,4) metallation pattern. Dimetallation is also found for the aldohexose phosphates. A mixed glycose-core-phosphate chelation was detected for Pd(II)(en) and M(III)(tacn) residues with M = Al, Ga in the pH range just above the physiological pH for the D-fructose 1,6-bisphosphate ligand. The results are discussed in relation to D-fructose-1,6-bisphosphate-metabolism in class-II aldolases. PMID:19771356

  3. Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

    DOE PAGES

    Niehaus, Thomas D.; Gerdes, Svetlana; Hodge-Hanson, Kelsey; Zhukov, Aleksey; Cooper, Arthur J.L.; ElBadawi-Sidhu, Mona; Fiehn, Oliver; Downs, Diana M.; Hanson, Andrew D.

    2015-05-15

    It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions. Phylogenetic analysis divided the Rid family into a widely distributed,more » apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate. Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine

  4. Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

    SciTech Connect

    Niehaus, Thomas D.; Gerdes, Svetlana; Hodge-Hanson, Kelsey; Zhukov, Aleksey; Cooper, Arthur J.L.; ElBadawi-Sidhu, Mona; Fiehn, Oliver; Downs, Diana M.; Hanson, Andrew D.

    2015-05-15

    It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions. Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate. Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine

  5. Quantum mechanical study of the β- and δ-lyase reactions during the base excision repair process: application to FPG.

    PubMed

    Sowlati-Hashjin, Shahin; Wetmore, Stacey D

    2015-10-14

    Bacterial FPG (or MutM) is a bifunctional DNA glycosylase that is primarily responsible for excising 8-oxoguanine (OG) from the genome by cleaving the glycosidic bond and the DNA backbone at the 3'- and 5'-phosphates of the damaged nucleoside. In the present work, quantum mechanical methods (SMD-M06-2X/6-311+G(2df,2p)//IEF-PCM-B3LYP/6-31G(d)) and a ring-opened Schiff base model that includes both the 3'- and 5'-phosphate groups are used to investigate the β- and δ-elimination reactions facilitated by FPG. Both the β- and δ-elimination reactions are shown to proceed through an E1cB mechanism that involves proton abstraction prior to the phosphate-ribose bond cleavage. Since transition states for the phosphate elimination reactions could not be characterized in the absence of leaving group protonation, our work confirms that the phosphate elimination reactions require protonation by a residue in the FPG active site, and can likely be further activated by additional active-site interactions. Furthermore, our model suggests that 5'-PO4 activation may proceed through a nearly isoenergetic direct (intramolecular) proton transfer involving the O4' proton of the deoxyribose of the damaged nucleoside. Regardless, our model predicts that both 3'- and 5'-phosphate protonation and elimination steps occur in a concerted reaction. Most importantly, our calculated barriers for the phosphate cleavage reactions reveal inherent differences between the β- and δ-elimination steps. Indeed, our calculations provide a plausible explanation for why the δ-elimination rather than the β-elimination is the rate-determining step in the BER facilitated by FPG, and why some bifunctional glycosylases (including the human counterpart, hOgg1) lack δ-lyase activity. Together, the new mechanistic features revealed by our work can be used in future large-scale modeling of the DNA-protein system to unveil the roles of key active sites residues in these relatively unexplored BER steps.

  6. The Prebiotic Chemistry of Nucleotides

    NASA Astrophysics Data System (ADS)

    Ferris, J. P.; Yanagawa, H.; Hagan, W. J., Jr.

    1984-12-01

    Diiminosuccinonitrile (DISN), formed by the oxidation of diaminomaleonitrile (DAMN), has been investigated as a potential prebiotic phosphorylating agent. DISN effects the cyclization of 3'-adenosine monophosphate to adenosine 2', 3'-cyclic phosphate in up to 39% yield. The mechanism of this reaction was investigated. The DISN-mediated phosphorylation of uridine to uridine monophosphate does not proceed efficiently in aqueous solution. The reaction of DISN with uridine-5'-phosphate and uridine results in the formation of 2,2'-anhydronucleotides and 2,2'-anhydronucleosides respectively, and other reaction products resulting from an initial reaction at the 2'- and 3'-hydroxyl groups. The clay mineral catalysis of the cyclization of adenosine-3'-phosphate was investigated using homoionic montmorillonites.

  7. Prebiotic synthesis and reactions of nucleosides and nucleotides

    NASA Astrophysics Data System (ADS)

    Ferris, J. P.; Yanagawa, H.; Hagan, W. J.

    Diiminosuccinonitrile (DISN) has been investigated as a potential prebiotic phosphorylating agent. It is formed readily by the oxidation of diaminomaleonitrile (DAMN), a tetramer of HCN, DISN effects the cyclization of 3'-adenosine monophosphate to adenosine 2',3'-cyclic phosphate in up to 40% yield. The DISN-mediated phosphorylation of uridine to uridine monophosphate does not proceed efficiently in aqueous solution. The reaction of DISN and BrCN with uridine-5'-phosphate and uridine results in the formation of 2,2'-anhydronucleotides and 2,2'-anhydronucleosides respectively, and other reaction products resulting from an initial reaction at the 2'- and 3'-hydroxyl groups. The clay mineral catalysis of the cyclization of adenosine-3'-phosphate was investigated using homoionic montmorillonites.

  8. Mechanism-based Inactivation by Aromatization of the Transaminase BioA Involved in Biotin Biosynthesis in Mycobaterium tuberculosis

    SciTech Connect

    Shi, Ce; Geders, Todd W.; Park, Sae Woong; Wilson, Daniel J.; Boshoff, Helena I.; Abayomi, Orishadipe; Barry, III, Clifton E.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2011-11-16

    BioA catalyzes the second step of biotin biosynthesis, and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5'-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 {angstrom} resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression, and these results provide support for the designed mechanism of action.

  9. Structural dynamics of a methionine γ-lyase for calicheamicin biosynthesis: Rotation of the conserved tyrosine stacking with pyridoxal phosphate.

    PubMed

    Cao, Hongnan; Tan, Kemin; Wang, Fengbin; Bigelow, Lance; Yennamalli, Ragothaman M; Jedrzejczak, Robert; Babnigg, Gyorgy; Bingman, Craig A; Joachimiak, Andrzej; Kharel, Madan K; Singh, Shanteri; Thorson, Jon S; Phillips, George N

    2016-05-01

    CalE6 from Micromonospora echinospora is a (pyridoxal 5' phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation. PMID:27191010

  10. Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins.

    PubMed

    Grin, Inga R; Khodyreva, Svetlana N; Nevinsky, Georgy A; Zharkov, Dmitry O

    2006-09-01

    Base excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5'-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase beta (Polbeta). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by beta-elimination with the efficiency similar to Polbeta. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polbeta. Our results suggest a role for NEILs as backup dRPases in mammalian cells.

  11. Pulse radiolysis studies of the interactions of the sulfhydryl compound dithiothreitol and sugars

    SciTech Connect

    Held, K.D.; Harrop, H.A.; Michael, B.D.

    1985-08-01

    Pulse radiolysis studies of the hydrogen atom transfer (repair) reaction from the sulfhydryl-containing (RSH) compound dithiothreitol (DTT) to the DNA sugar deoxyribose and to several related sugars have been undertaken. The H transfer reaction is measured by monitoring the transient absorbance of the radical-anion RSSR/sup -/. The H atom transfer reactions for some sugars were fitted by a single time exponential function, but other sugars exhibited both a fast and a slow component to the reaction. The maximum extent of total repair varied from 60% for ribose-5-phosphate to 100% for 2-deoxyglucose. The rate of repair, the extent of repair, and the appearance of more than one component of repair seem to depend on several factors. The biological relevance of the reactions studied herein is discussed and the rates obtained are compared with rates for repair of damage in certain radiobiological systems.

  12. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water

    PubMed Central

    Cafferty, Brian J.; Fialho, David M.; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V.

    2016-01-01

    The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life. PMID:27108699

  13. Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Wu, Chao; Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Wen-Wei; Li, Dao-Bo; Yu, Han-Qing

    2013-05-01

    Shewanella oneidensis MR-1 is an extensively studied dissimilatory metal-reducing bacterium with a great potential for bioremediation and electricity generation. It secretes flavins as electron shuttles which play an important role in extracellular electron transfer. However, the influence of various environmental factors on the secretion of flavins is largely unknown. Here, the effects of electron acceptors, including fumarate, ferrihydrite, Fe(III)-nitrilotriacetic acid (NTA), nitrate and trimethylamine oxide (TMAO), on the secretion of flavins were investigated. The level of riboflavin and riboflavin-5'-phosphate (FMN) secreted by S. oneidensis MR-1 varied considerably with different electron acceptors. While nitrate and ferrihydrite suppressed the secretion of flavins in relative to fumarate, Fe(III)-NTA and TMAO promoted such a secretion and greatly enhanced ferrihydrite reduction and electricity generation. This work clearly demonstrates that electron acceptors could considerably affect the secretion of flavins and consequent microbial EET. Such impacts of electron acceptors in the environment deserve more attention.

  14. The folding characteristics of tryptophanase from Escherichia coli.

    PubMed

    Mizobata, T; Kawata, Y

    1995-02-01

    The unfolding and refolding characteristics of Escherichia coli tryptophanase (tryptophan indole-lyase) [EC 4.1.99.1] in guanidine hydrochloride were studied. Tryptophanase unfolded by first dissociating its coenzyme, pyridoxal 5'-phosphate, from the active site. This dissociation caused a significant destabilization of structure, and global unfolding of the protein followed. During this global unfolding step, an intermediate was formed which had a strong tendency to aggregate irreversibly, as detected by light scattering experiments. Tryptophanase was unable to refold quantitatively after unfolding in 4 M guanidine hydrochloride. The low refolding yield was due to non-specific aggregation which occurs during refolding. Various conditions which limited this aggregation were probed, and it was found that by initiating the refolding reaction at low temperature, the aggregation of tryptophanase folding intermediates during the reaction could be avoided to a certain extent, and the refolding yield improved.

  15. Rational Design, Synthesis, and Preliminary Structure-Activity Relationships of α-Substituted-2-Phenylcyclopropane Carboxylic Acids as Inhibitors of Salmonella typhimurium O-Acetylserine Sulfhydrylase.

    PubMed

    Pieroni, Marco; Annunziato, Giannamaria; Beato, Claudia; Wouters, Randy; Benoni, Roberto; Campanini, Barbara; Pertinhez, Thelma A; Bettati, Stefano; Mozzarelli, Andrea; Costantino, Gabriele

    2016-03-24

    Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5'-phosphate (PLP)-dependent enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of cysteine, OASS exerts a series of "moonlighting" activities in bacteria, such as transcriptional regulation, contact-dependent growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations.

  16. Crystallization and preliminary X-ray analysis of a phosphopentomutase from Bacillus cereus

    SciTech Connect

    Panosian, Timothy D.; Nannemann, David P.; Bachmann, Brian O.; Iverson, T.M.

    2013-09-18

    Phosphopentomutases (PPMs) interconvert D-ribose 5-phosphate and {alpha}-D-ribose 1-phosphate to link glucose and nucleotide metabolism. PPM from Bacillus cereus was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Bacterial PPMs are predicted to contain a di-metal reaction center, but the catalytically relevant metal has not previously been identified. Sparse-matrix crystallization screening was performed in the presence or absence of 50 mM MnCl{sub 2}. This strategy resulted in the formation of two crystal forms from two chemically distinct conditions. The crystals that formed with 50 mM MnCl{sub 2} were more easily manipulated and diffracted to higher resolution. These results suggest that even if the catalytically relevant metal is not known, the crystallization of putative metalloproteins may still benefit from supplementation of the crystallization screens with potential catalytic metals.

  17. 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

  18. Amenable Treatable Severe Pediatric Epilepsies.

    PubMed

    Pearl, Phillip L

    2016-05-01

    Vitamin-dependent epilepsies and multiple metabolic epilepsies are amenable to treatment that markedly improves the disease course. Knowledge of these amenably treatable severe pediatric epilepsies allows for early identification, testing, and treatment. These disorders present with various phenotypes, including early onset epileptic encephalopathy (refractory neonatal seizures, early myoclonic encephalopathy, and early infantile epileptic encephalopathy), infantile spasms, or mixed generalized seizure types in infancy, childhood, or even adolescence and adulthood. The disorders are presented as vitamin responsive epilepsies such as pyridoxine, pyridoxal-5-phosphate, folinic acid, and biotin; transportopathies like GLUT-1, cerebral folate deficiency, and biotin thiamine responsive disorder; amino and organic acidopathies including serine synthesis defects, creatine synthesis disorders, molybdenum cofactor deficiency, and cobalamin deficiencies; mitochondrial disorders; urea cycle disorders; neurotransmitter defects; and disorders of glucose homeostasis. In each case, targeted intervention directed toward the underlying metabolic pathophysiology affords for the opportunity to significantly effect the outcome and prognosis of an otherwise severe pediatric epilepsy. PMID:27544473

  19. The oxidative pentose phosphate pathway in the haloarchaeon Haloferax volcanii involves a novel type of glucose-6-phosphate dehydrogenase--The archaeal Zwischenferment.

    PubMed

    Pickl, Andreas; Schönheit, Peter

    2015-04-28

    The oxidative pentose phosphate pathway (OPPP), catalyzing the oxidation of glucose-6-phosphate to ribulose-5-phosphate is ubiquitous in eukarya and bacteria but has not yet been reported in archaea. In haloarchaea a putative 6-phosphogluconate dehydrogenase (6PGDH) is annotated, whereas a gene coding for glucose-6-phosphate dehydrogenase (Glc6PDH) could not be identified. Here we report the purification and characterization of a novel type of Glc6PDH in Haloferax volcanii that is not related to bacterial and eukaryal Glc6PDHs and the encoding gene is designated as azf (archaeal zwischenferment). Further, recombinant H. volcanii 6PGDH was characterized. Deletion mutant analyses indicate that both, Glc6PDH and 6PGDH, are functionally involved in pentose phosphate formation in vivo. This is the first report on the operation of the OPPP in the domain of archaea.

  20. Redesigning Aldolase Stereoselectivity by Homologous Grafting.

    PubMed

    Bisterfeld, Carolin; Classen, Thomas; Küberl, Irene; Henßen, Birgit; Metz, Alexander; Gohlke, Holger; Pietruszka, Jörg

    2016-01-01

    The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity. PMID:27327271

  1. Alanine racemase from the acidophile Acetobacter aceti.

    PubMed

    Francois, Julie A; Kappock, T Joseph

    2007-01-01

    Acetobacter aceti converts ethanol to acetic acid, and survives acetic acid exposure by tolerating cytoplasmic acidification. Alanine racemase (Alr) is a pyridoxal 5' phosphate (PLP) -dependent enzyme that catalyzes the interconversion of the d- and l-isomers of alanine and has a basic pH optimum. Since d-alanine is essential for peptidoglycan biosynthesis, Alr must somehow function in the acidic cytoplasm of A. aceti. We report the partial purification of native A. aceti Alr (AaAlr) and evidence that it is a rather stable enzyme. The C-terminus of AaAlr has a strong resemblance to the ssrA-encoded protein degradation signal, which thwarted initial protein expression experiments. High-activity AaAlr forms lacking a protease recognition sequence were expressed in Escherichia coli and purified. Biophysical and enzymological experiments confirm that AaAlr is intrinsically acid-resistant, yet has the catalytic properties of an ordinary Alr.

  2. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis.

    PubMed

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T

    2015-01-01

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. PMID:26569229

  3. Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Wu, Chao; Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Wen-Wei; Li, Dao-Bo; Yu, Han-Qing

    2013-05-01

    Shewanella oneidensis MR-1 is an extensively studied dissimilatory metal-reducing bacterium with a great potential for bioremediation and electricity generation. It secretes flavins as electron shuttles which play an important role in extracellular electron transfer. However, the influence of various environmental factors on the secretion of flavins is largely unknown. Here, the effects of electron acceptors, including fumarate, ferrihydrite, Fe(III)-nitrilotriacetic acid (NTA), nitrate and trimethylamine oxide (TMAO), on the secretion of flavins were investigated. The level of riboflavin and riboflavin-5'-phosphate (FMN) secreted by S. oneidensis MR-1 varied considerably with different electron acceptors. While nitrate and ferrihydrite suppressed the secretion of flavins in relative to fumarate, Fe(III)-NTA and TMAO promoted such a secretion and greatly enhanced ferrihydrite reduction and electricity generation. This work clearly demonstrates that electron acceptors could considerably affect the secretion of flavins and consequent microbial EET. Such impacts of electron acceptors in the environment deserve more attention. PMID:23558182

  4. 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.

  5. Purification and Properties of Adenosine Diphosphoglucose Pyrophosphorylase from Sweet Corn 1

    PubMed Central

    Amir, Jacob; Cherry, Joe H.

    1972-01-01

    A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg2+ and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1-phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 × 10−4, 3.2 × 10−5, 3.3 × 10−5, and 6.2 × 10−4m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 × 10−7m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate. PMID:16658078

  6. Rational Design, Synthesis, and Preliminary Structure-Activity Relationships of α-Substituted-2-Phenylcyclopropane Carboxylic Acids as Inhibitors of Salmonella typhimurium O-Acetylserine Sulfhydrylase.

    PubMed

    Pieroni, Marco; Annunziato, Giannamaria; Beato, Claudia; Wouters, Randy; Benoni, Roberto; Campanini, Barbara; Pertinhez, Thelma A; Bettati, Stefano; Mozzarelli, Andrea; Costantino, Gabriele

    2016-03-24

    Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5'-phosphate (PLP)-dependent enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of cysteine, OASS exerts a series of "moonlighting" activities in bacteria, such as transcriptional regulation, contact-dependent growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations. PMID:26894308

  7. Activation and inactivation of methanol: 2-mercaptoethanesulfonic acid methyltransferase from Methanosarcina barkeri.

    PubMed Central

    van der Meijden, P; Heythuysen, H J; Sliepenbeek, H T; Houwen, F P; van der Drift, C; Vogels, G D

    1983-01-01

    Methanol is converted to methane by crude extracts of Methanosarcina barkeri. The first reaction involved in this process, is catalyzed by methanol:2-mercaptoethanesulfonic acid methyltransferase (EC 2.1.1.-). The methyltransferase has an optimum at pH 6.5 and is not inhibited by 2-bromoethanesulfonic acid. Pyridoxal-5'-phosphate acts as an inhibitor (Ki = 0.30 mM). The methyltransferase was tested in the presence of 2-bromoethanesulfonic acid, which inhibits the conversion of 2-(methylthio)ethanesulfonic acid to methane. The reaction is subject to activation and inactivation. Inactivation is brought about by the presence of oxygen, flavin mononucleotide, flavin adenine dinucleotide, and 2-(methylthio)ethanesulfonic acid, the product of the reaction. Activation of the system requires the presence of ATP and Mg2+ and of hydrogen. Hydrogen can be replaced by enzymatic systems, such as pyruvate dehydrogenase, which deliver free hydrogen. PMID:6294063

  8. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

    PubMed

    Fenalti, Gustavo; Law, Ruby H P; Buckle, Ashley M; Langendorf, Christopher; Tuck, Kellie; Rosado, Carlos J; Faux, Noel G; Mahmood, Khalid; Hampe, Christiane S; Banga, J Paul; Wilce, Matthew; Schmidberger, Jason; Rossjohn, Jamie; El-Kabbani, Ossama; Pike, Robert N; Smith, A Ian; Mackay, Ian R; Rowley, Merrill J; Whisstock, James C

    2007-04-01

    Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

  9. Endogenous ethanol--its metabolic, behavioral and biomedical significance.

    PubMed

    Ostrovsky YuM

    1986-01-01

    Ethanol is constantly formed endogenously from acetaldehyde, and level of the former can be measured in both human beings and animals. Acetaldehyde can be generated in situ from the metabolism of pyruvate, threonine, deoxyribose-5-phosphate, phosphoethanolamine, alanine and presumably from other substrates. The levels of blood and tissue endogenous ethanol change as a function of various physiologic and experimental conditions such as starvation, aging, stress, cooling, adrenalectomy, etc. and are regulated by many exogenous compounds such as antimetabolites, derivatives of amino acids, lithium salts, disulfiram, cyanamide, etc. Under free choice alcohol selection situations, the levels of endogenous ethanol in rat blood and alcohol preference by the animals are negatively correlated. Similar negative correlations have been found between the levels of blood endogenous ethanol and the frequency of delirium in alcoholic patients undergoing alcohol withdrawal. Endogenous ethanol and acetaldehyde can therefore be regarded as compounds which fulfil substrate, regulatory and modulator functions.

  10. Substrate inhibition of transketolase.

    PubMed

    Solovjeva, Olga N; Kovina, Marina V; Kochetov, German A

    2016-03-01

    We studied the influence of the acceptor substrate of transketolase on the activity of the enzyme in the presence of reductants. Ribose-5-phosphate in the presence of cyanoborohydride decreased the transketolase catalytic activity. The inhibition is caused by the loss of catalytic function of the coenzyme-thiamine diphosphate. Similar inhibitory effect was observed in the presence of NADPH. This could indicate its possible regulatory role not only towards transketolase, but also towards the pentose phosphate pathway of carbohydrate metabolism overall, taking into account the fact that it inhibits not only transketolase but also another enzyme of the pentose phosphate pathway--glucose 6-phosphate dehydrogenase [Eggleston L.V., Krebs H.A. Regulation of the pentose phosphate cycle, Biochem. J. 138 (1974) 425-435]. PMID:26708478

  11. Dual isotope labeling: conjugation of 32P-oligonucleotides with 18F-aryltrifluoroborate via copper(I) catalyzed cycloaddition.

    PubMed

    Li, Ying; Schaffer, Paul; Perrin, David M

    2013-12-01

    A one-pot-two-step labeling of an oligonucleotide with an (18)F-ArBF3(-)(aryltrifluoroborate) radioprosthetic is reported herein. In order to characterize labeling in terms of radiochemistry, phosphorus-32 was also introduced to the 5'-terminus of the oligonucleotide via enzymatic phosphorylation. A pendant azide group was subsequently conjugated to the 5'-phosphate of the oligonucleotide. Copper(I) catalyzed [2+3] cycloaddition was undertaken to conjugate an alkyne-bearing(18)F-ArBF3(-) to the oligonucleotide. Following polyacrylamide gel electrophoresis, this doubly-labeled bioconjugate exhibited decay properties of both the phosphorus-32 and fluorine-18, that were confirmed by autoradiography at selected lengths of time, which in turn provided concrete evidence of successful conjugation. These results are corroborated by HPLC analysis of the labeled material. Taken together this work demonstrates viable use of (18)F-ArBF3(-) prosthetics for labeling oligonucleotides for use in PET imaging. PMID:24144852

  12. Formation of nucleoside 5'-polyphosphates from nucleotides and trimetaphosphate

    NASA Technical Reports Server (NTRS)

    Lohrmann, R.

    1975-01-01

    Nucleoside 5'-polyphosphates (N5PP) formed when solutions of nucleoside 5'-phosphates (N5P) and trimetaphosphate (TMP) are dessicated at room temperature are studied by paper chromatography, electrophoresis, and metal catalytic reactions. Divalent Mg ion exhibited superior catalytic function to other divalent metal ions in the reaction. Major reaction products are indicated. The importance of the N5PP series, TMP, and N5-triphosphate as substrates of RNA and DNA synthesis, and under postulated prebiotic conditions likely to obtain during prebiological ages of the earth, is emphasized and discussed. Alternate drying and wetting, evaporation from a prebiotic puddle, concentration of solubles in the remaining liquid phase, metal catalysis, and the role of these substances in the formation of amino acids and long-chain polyphosphates are considered.

  13. Membrane-associated Sulfur Oxidation by the Autotroph Thiobacillus thiooxidans

    PubMed Central

    Adair, Frank W.

    1966-01-01

    Adair, Frank W. (Rutgers, The State University, New Brunswick, N. J.). Membrane-associated sulfur oxidation by the autotroph Thiobacillus thiooxidans. J. Bacteriol. 92:899–904. 1966.—Washed cell wall-membrane fragments derived from sulfur-grown cells of the strictly autotrophic bacterium, Thiobacillus thiooxidans, oxidized elemental sulfur to sulfate without the addition of cofactors. The oxidation was optimal at pH 7.0 and was increased by the presence of wetting agents. Oxygen uptake was inhibited by cyanide, azide, and thiol-binding agents. Sulfite was also oxidized, and both the sulfur- and sulfite-oxidizing systems were heat-labile. Neither thiosulfate nor tetrathionate was oxidized by soluble or membrane preparations. The fragments fixed C14O2 in the presence of ribose-5-phosphate, Mg++, and adenosine triphosphate. Sulfur oxidation did not provide energy for C14O2 fixation in this system. PMID:5926757

  14. Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase.

    PubMed

    Volk, R; Bacher, A

    1990-11-15

    The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-181). The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures. The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa. The committed substrate of the enzyme was identified as D-ribulose 5-phosphate. The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy. Mg2+ is required for activity. PMID:2246238

  15. Redesigning Aldolase Stereoselectivity by Homologous Grafting

    PubMed Central

    Henßen, Birgit; Metz, Alexander; Gohlke, Holger; Pietruszka, Jörg

    2016-01-01

    The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity. PMID:27327271

  16. Controlling reaction specificity in pyridoxal phosphate enzymes

    PubMed Central

    Toney, Michael D.

    2012-01-01

    Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at Cα of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. PMID:21664990

  17. [Ion-pair HPLC analysis of B vitamins in syrup products in Indonesia].

    PubMed

    Miyazaki, Tamaki; Horisaki, Tadafumi; Aso, Yukio; Okuda, Haruhiro

    2013-01-01

    A training course for analysis of B vitamins in syrup products was undertaken at the National Agency of Drug and Food Control at Jakarta as part of the project to deliver safe drugs to people in Indonesia by Japan International Cooperation Agency. Analytical methods have been developed for quantitative determination of B vitamins by ion-pair high-performance liquid chromatography using 1-hexanesulfonic acid sodium salt. Measurements were performed for two syrup products removed from a drug store in Jakarta to determine the amount of each vitamin B. The measured values of riboflavin 5'-phosphate sodium, nicotinamide and pyridoxine hydrochloride were almost the same with those of nominal content for both products. While the measured values of thiamine hydrochloride, pantothenol and cyanocobalamin were approximately twice the amount of nominal contents.

  18. Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits.

    PubMed

    Whitman, W B; Martin, M N; Tabita, F R

    1979-10-25

    Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.

  19. [Synthesis of D-ribulose-1,5-diphosphate with immobilized enzymes of Thiobacillus].

    PubMed

    Khaga, M E; Mikel'saar, P Ch; Liaene, A E; Aaviksaar, A A; Peenema, E V

    1979-01-01

    Preparative synthesis of D-ribulose-1,5-diphosphate (RuDP) from ribose-5-phosphate and ATP was carried out, using as a catalyst a crude extract of Thiobacillus thiooxidans 58 R immobilized on porous glass. The methods for immobiliztion of crude bacterial extracts, synthesis of RuDP and purification of the resultant product by means of column chromatography on activated charcoal and anionites were developed. The structure of RuDP was identified by 13C-NMR spectroscopy. Stability of two phosphate groups of RuDP during acid and alkaline hydrolysis proved to be different: both phosphate groups were completely removed in 1 N H2SO4 at 100 degrees C whereas only one phosphate group was hydrolysed in 1 N NaOH at 25 degrees C. This finding is at variance with the earlier results of Horecker et al. (1956).

  20. DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

    NASA Astrophysics Data System (ADS)

    Harper, Brett; Neumann, Elizabeth K.; Solouki, Touradj

    2015-08-01

    Collision-induced dissociation (CID) of m/z-isolated w type fragment ions and an intact 5' phosphorylated DNA oligonucleotide generated rearranged product ions. Of the 21 studied w ions of various nucleotide sequences, fragment ion sizes, and charge states, 18 (~86%) generated rearranged product ions upon CID in a Synapt G2-S HDMS (Waters Corporation, Manchester, England, UK) ion mobility-mass spectrometer. Mass spectrometry (MS), ion mobility spectrometry (IMS), and theoretical modeling data suggest that purine bases can attack the free 5' phosphate group in w type ions and 5' phosphorylated DNA to generate sequence permuted [phosphopurine]- fragment ions. We propose and discuss a potential mechanism for generation of rearranged [phosphopurine]- and complementary y-B type product ions.

  1. Phosphate sensor based on immobilized aluminium-morin in poly (glycidyl methacrylate) microspheres

    NASA Astrophysics Data System (ADS)

    Ahmad, Amalina; Hanifah, Sharina Abu; Hasbullah, Siti Aishah; Suhud, Khairi; Zaini, Norhadisah Mohd; Heng, Lee Yook

    2014-09-01

    This paper reports the development of dihydrogen phosphate ion (H2PO4-) sensor in free solution and immobilized aluminium-morin (Al-Mo) complex on poly(glycidyl methacrylate) (pGMA) microspheres. The immobilization was carried out by suspension photopolymerization technique. Based on Al-Mo solution work, phosphate can be detected from 0.1 - 15.0 ppm of dihydrogen phosphate at pH 5. Phosphate detection only takes about 5 minutes. Morphology analyses showed that the immobilization of Aluminium-Morin complex maintained the size of the microspheres and proved that entrapment involves in the formation of the microspheres. This result is further explained by Attenuated Total Reflectance (ATR) spectrum which does not show any formation of new bands. The microspheres were then used for further applications.

  2. Synthesis of amino acyl adenylates using the tert-butoxycarbonyl protecting group

    NASA Technical Reports Server (NTRS)

    Armstrong, D. W.; Seguin, R.; Saburi, M.; Fendler, J. H.

    1979-01-01

    The synthesis of amino acyl adenylates using N-tert-butoxycarbonyl-protected amino acids is reported. Anhydrous solutions containing N-tert-butoxycarbonyl alanine, phenylalanine, and methionine were combined with the anhydrous mono (tri-n-octylammonium) salt of adenosine 5'-phosphate and the resultant amino acyl adenylates were characterized by means of elemental analysis, and infrared and proton NMR spectroscopy. Amino acyl adenylate yields of up to 60% were obtained with high purity at room temperatures. The reported synthesis is considered to represent a large improvement over previous methods due to the purity of the products, normal temperature requirements, and the stability of the starting compounds, which suggests its use in investigations of prebiotic oligo- and polypeptide synthesis.

  3. Analysis of a new cluster of genes involved in the synthesis of the unique volatile organic compound sodorifen of Serratia plymuthica 4Rx13.

    PubMed

    Domik, Dajana; Magnus, Nancy; Piechulla, Birgit

    2016-07-01

    The rhizobacterium Serratia plymuthica 4Rx13 emits the novel and unique volatile sodorifen (C16H26), which has a polymethylated bicyclic structure. Transcriptome analysis revealed that gene SOD_c20750 (annotated as terpene cyclase) is involved in the biosynthesis of sodorifen. Here we show that this gene is located in a small cluster of four genes (SOD_c20750 - SOD_c20780), and the analysis of the knockout mutants demonstrated that SOD_c20760 (annotated as methyltransferase) and SOD_c20780 (annotated as isopentenyl pyrophosphate (IPP) isomerase) are needed for the biosynthesis of sodorifen, while a sodorifen-negative phenotype was not achieved with the SOD_c20770 (annotated as deoxy-xylulose-5-phosphate (DOXP) synthase) mutant. Altogether, the function of this new gene cluster was assigned to the biosynthesis of this structurally unusual volatile compound sodorifen.

  4. Plastid-cytosol partitioning and integration of metabolic pathways for APS/PAPS biosynthesis in Arabidopsis thaliana.

    PubMed

    Bohrer, Anne-Sophie; Kopriva, Stanislav; Takahashi, Hideki

    2014-01-01

    Plants assimilate sulfate from the environment to synthesize biologically active sulfur-containing compounds required for growth and cellular development. The primary steps of sulfur metabolism involve sequential enzymatic reactions synthesizing adenosine 5'-phosphosulfate (APS) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Recent finding suggests that an adenosine nucleotide transport system facilitating the exchange of PAPS and 3'-phosphoadenosine 5'-phosphate across the plastid envelope is essential for establishing an intimate connection between the plastidic and cytosolic sulfate assimilation pathways in plants. Subcellular partitioning and integration of metabolic pathways provide focal points for investigating metabolic flux regulations. This perspective article presents an integrative view of sulfur metabolic flux control mechanisms with an emphasis on subcellular partitioning of APS/PAPS biosynthetic pathways in Arabidopsis thaliana.

  5. Sensitive and rapid determination of quinoline yellow in drinks using polyvinylpyrrolidone-modified electrode.

    PubMed

    Zhang, Shenghui; Shi, Zhen; Wang, Jinshou

    2015-04-15

    A novel electrochemical sensor using polyvinylpyrrolidone (PVP)-modified carbon paste electrode was developed for the sensitive and rapid determination of quinoline yellow. In 0.1M, pH 6.5 phosphate buffer, an irreversible oxidation wave at 0.97 V was observed for quinoline yellow. PVP exhibited strong accumulation ability to quinoline yellow, and consequently increased the oxidation peak current of quinoline yellow remarkably. The effects of pH value, amount of PVP, accumulation potential and time were studied on the oxidation signals of quinoline yellow. The linear range was from 5×10(-8) to 1×10(-6) M, and the limit of detection was evaluated to be 2.7×10(-8) M. It was used to detect quinoline yellow in different drink samples, and the results consisted with the values that obtained by high-performance liquid chromatography.

  6. Crystal Structures of the Glycopeptide Sulfotransferase Teg12 Complexed with the Teicoplanin Aglycone

    PubMed Central

    Bick, Matthew J.; Banik, Jacob J.; Darst, Seth A.; Brady, Sean F.

    2010-01-01

    The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, 13, and 14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures including: an apo structure of Teg12, Teg12 bound to the desulfated co-substrate 3'-phosphoadenosine 5'-phosphate and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions. PMID:20361791

  7. Serum concentrations of micronutrients, packed cell volume, and blood hemoglobin during the first two gestations and lactations of sows.

    PubMed Central

    Girard, C L; Robert, S; Matte, J J; Farmer, C; Martineau, G P

    1996-01-01

    The objective of the present work was to describe the changes in serum concentrations of some micronutrients during the first 2 gestations and lactations of 33 gilts in order to establish blood reference values for a rapid assessment of nutritional status. In both parities, blood samples were taken from the jugular vein at mating, 5, 10 and 15 wk of gestation and l d and 4 wk after parturition (weaning). Reference values (mean, standard deviation, minimum, maximum) for serum folates, vitamin B12, vitamin B6 metabolites (pyridoxal and pyridoxal-5-phosphate), calcium, phosphorus, sodium, zinc, copper and iron, as well as blood hemoglobin and packed cell volume are reported for each studied time. Differences between parities and between each time are also reported. Results from the present report demonstrate that knowledge of the physiological state of the sows is critical for the assessment of nutritional status of an individual or a breeding herd by interpretation of analyses of blood constituents. PMID:8809380

  8. Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate

    SciTech Connect

    Sharma, O.K.; Goswami, B.B.

    1981-04-01

    Extracts of interferon-treated cells synthesize unique 2',5'-linked oligo(adenylic acid) 5'-phosphates in the presence of ATP and double-stranded RNA. 2',5'-linked oligo(adenylic acid) 5'-triphosphate inhibits protein synthesis at nanomolar concentrations by activating RNase. We have observed that oligo(adenylic acid) 5'-monophosphate and 5'-triphosphate are potent inhibitors of vaccinia mRNA methylation in vitro. Both the methylation of the 5'-terminal guanine at the 7 position and the 2'-O-ribose methylation of the penultimate nucleoside are inhibited. Such inhibition of mRNA methylation is not due to degradation of the mRNA. Inhibition of the requisite modification of the 5' terminus of mRNA by 2',5'-linked oligo(adenylic acids) may be a mechanism of interferon action against both DNA and RNA viruses in which mRNAs derived from them are capped.

  9. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water.

    PubMed

    Cafferty, Brian J; Fialho, David M; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V

    2016-01-01

    The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life. PMID:27108699

  10. Trading off stability against activity in extremophilic aldolases

    PubMed Central

    Dick, Markus; Weiergräber, Oliver H.; Classen, Thomas; Bisterfeld, Carolin; Bramski, Julia; Gohlke, Holger; Pietruszka, Jörg

    2016-01-01

    Understanding enzyme stability and activity in extremophilic organisms is of great biotechnological interest, but many questions are still unsolved. Using 2-deoxy-D-ribose-5-phosphate aldolase (DERA) as model enzyme, we have evaluated structural and functional characteristics of different orthologs from psychrophilic, mesophilic and hyperthermophilic organisms. We present the first crystal structures of psychrophilic DERAs, revealing a dimeric organization resembling their mesophilic but not their thermophilic counterparts. Conversion into monomeric proteins showed that the native dimer interface contributes to stability only in the hyperthermophilic enzymes. Nevertheless, introduction of a disulfide bridge in the interface of a psychrophilic DERA did confer increased thermostability, suggesting a strategy for rational design of more durable enzyme variants. Constraint network analysis revealed particularly sparse interactions between the substrate pocket and its surrounding α-helices in psychrophilic DERAs, which indicates that a more flexible active center underlies their high turnover numbers. PMID:26783049

  11. One-pot microbial synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase.

    PubMed

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-06-01

    A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2'-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2'-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.

  12. A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation.

    PubMed Central

    Tuschl, T; Sharp, P A; Bartel, D P

    2001-01-01

    In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation. PMID:11214178

  13. Fracture of solid state laser slabs

    SciTech Connect

    Marion, J.E.

    1986-07-01

    Fracture due to thermal stress limits the power output potential of modern, high average power slab lasers. Here the criteria for slab fracture and the nature of the surface flaws which constitute the strength-controlling defects are reviewed. Specific fracture data for gadolinium scandium gallium garnet and LHG-5 phosphate glass with different surface finishes are evaluated in the context of assigning appropriate slab operating parameters using Wiebull statistics. These examples illustrate both the danger of design using brittle components without adequate fracture testing, and the inadequacy of design methods which use a fixed safety factor, for this class of materials. Further consideration reveals that operation of slab lasers in contact with an aqueous coolant may lead to strength degradation with time. Finally, the evolution of the failure process in which a characteristic midplane crack forms is outlined, and the pertinent parameters for avoiding slab fracture are identified.

  14. A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway.

    PubMed

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J

    2006-10-01

    Plants are able to integrate exogenous 1-deoxy-D-xylulose (DX) into the 2C-methyl-D-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-D-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing D-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as "xylulose kinase" might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.

  15. A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

    PubMed Central

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

    2006-01-01

    Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

  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. Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S ,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

    SciTech Connect

    Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I.; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-23

    γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.

  18. Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S ,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

    DOE PAGES

    Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I.; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-23

    γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

  19. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    PubMed

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations. PMID:21621574

  20. Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases.

    PubMed

    Chen, Zan; Thomas, Stefani N; Bolduc, David M; Jiang, Xuejun; Zhang, Xiangbin; Wolberger, Cynthia; Cole, Philip A

    2016-07-01

    PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN's C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN's cellular stability.

  1. Use of phosphoimidazolide-activated guanosine to investigate the nucleophilicity of spermine and spermidine

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Baird, E. E.; Smith, P. J.

    1995-01-01

    Guanosine 5'-phosphate 2-methylimidazolide (2-MeImpG), a labile phosphoimidazolide analog of guanosine triphosphate, was used to test the reactivity of the natural polyamines (PAs), spermine (spm) and spermidine (spd). The products are the guanosine 5'-phosphate-polyamine derivatives (PA-pG: spd-pG and spm-pG) which are quite stable in the range 4 < pH < 11. Our study is the first of which we are aware that reports on the nucleophilicity of these amines. The main findings are as follows. (i) HPLC analysis of the products indicates the formation of only two of the three possible spd products and only one of the two possible spm products. These results can be explained if only the primary amino groups of the two polyamines are reactive, while the secondary amino groups are rendered unreactive by a steric effect. The reactions of 2-MeImpG and other phosphoimidazolide derivatives of nucleosides (ImpNs) with primary and secondary monoamines support this interpretation (Kanavarioti et al. J. Org. Chem. 1995, 60, 632). (ii) The product ratio of the two spd-pG adducts derived from the primary amino groups varies between 2.40 and 0.71 in the range 6.1 < or equal to pH < or equal to 11.9. Such small variation in the product ratio can only be rationalized by the similar, but not identical, basicity of the two primary amino groups and provides strong support for a previously reported model for polyamine ionization (Onasch et. al. Biophys. Chem. 1984, 19, 245). (iii) On the basis of our kinetic determinations conditions at which the nucleophilicity of these amines is at a minimum and at which other interactions with ImpNs could be tested can be chosen.

  2. The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane

    PubMed Central

    Strøm, Terje; Ferenci, Thomas; Quayle, J. Rodney

    1974-01-01

    d-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C1 assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested. PMID:4377654

  3. High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase.

    PubMed

    Schwartz, H L; Chandonia, J M; Kash, S F; Kanaani, J; Tunnell, E; Domingo, A; Cohen, F E; Banga, J P; Madec, A M; Richter, W; Baekkeskov, S

    1999-04-16

    The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells. PMID:10222205

  4. A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation.

    PubMed

    Negi, Vishal Singh; Bingham, Jon-Paul; Li, Qing X; Borthakur, Dulal

    2014-02-01

    The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future. PMID:24351687

  5. Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis.

    PubMed

    Papini, Marta; Nookaew, Intawat; Siewers, Verena; Nielsen, Jens

    2012-08-01

    Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.

  6. Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.

    PubMed

    Soo, Valerie W C; Yosaatmadja, Yuliana; Squire, Christopher J; Patrick, Wayne M

    2016-09-16

    Enzymes that utilize the cofactor pyridoxal 5'-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine β-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine β-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat ≈10 h(-1)). In contrast, directed evolution also yielded CBL(P113S), which catalyzes l-alanine racemization with a poor Km (58 mm) but a high kcat (22 s(-1)). The structures of both variants were solved in the presence and absence of the l-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during l-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5'-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor. PMID:27474741

  7. HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatase AtSgpp.

    PubMed

    Caparrós-Martín, José A; McCarthy-Suárez, Iva; Culiáñez-Macià, Francisco A

    2013-04-01

    This work presents the isolation and the biochemical characterization of the Arabidopsis thaliana gene AtSgpp. This gene shows homology with the Arabidopsis low molecular weight phosphatases AtGpp1 and AtGpp2 and the yeast counterpart GPP1 and GPP2, which have a high specificity for DL-glycerol-3-phosphate. In addition, it exhibits homology with DOG1 and DOG2 that dephosphorylate 2-deoxy-D-glucose-6-phosphate. Using a comparative genomic approach, we identified the AtSgpp gene as a conceptual translated haloacid dehalogenase-like hydrolase HAD protein. AtSgpp (locus tag At2g38740), encodes a protein with a predicted Mw of 26.7 kDa and a pI of 4.6. Its sequence motifs and expected structure revealed that AtSgpp belongs to the HAD hydrolases subfamily I, with the C1-type cap domain. In the presence of Mg(2+) ions, the enzyme has a phosphatase activity over a wide range of phosphosugars substrates (pH optima at 7.0 and K m in the range of 3.6-7.7 mM). AtSgpp promiscuity is preferentially detectable on D-ribose-5-phosphate, 2-deoxy-D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-fructose-1-phosphate, D-glucose-6-phosphate, DL-glycerol-3-phosphate, and D-fructose-6-phosphate, as substrates. AtSgpp is ubiquitously expressed throughout development in most plant organs, mainly in sepal and guard cell. Interestingly, expression is affected by abiotic and biotic stresses, being the greatest under Pi starvation and cyclopentenone oxylipins induction. Based on both, substrate lax specificity and gene expression, the physiological function of AtSgpp in housekeeping detoxification, modulation of sugar-phosphate balance and Pi homeostasis, is provisionally assigned.

  8. The multiple Maillard reactions of ribose and deoxyribose sugars and sugar phosphates.

    PubMed

    Munanairi, Admire; O'Banion, Steven K; Gamble, Ryan; Breuer, Elizabeth; Harris, Andrew W; Sandwick, Roger K

    2007-12-10

    Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sugars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reaction, provides the opportunity for the R5P molecule to undergo novel reaction paths creating unique Maillard products. The initial set of reactions leading to an Amadori product (phosphorylated) and to an alpha-dicarbonyl phosphate compound follows a typical Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the products formed. The reaction rate for the loss of R5P is partially dependent on the pK(a) of the amine but also is correlated to the protonation of an early intermediate of the reaction sequence. In the presence of oxygen, a carboxymethyl group conjugated to the amine is a major product of the reaction of R5P with N-acetyllysine while little of this product is generated in the absence of oxygen. Despite lacking a critical hydroxyl group necessary for the Maillard reaction, 2-deoxyribose 5-phosphate (dR5P) still generates an Amadori-like product (with a carbonyl on the C-3 carbon) and undergoes phosphate cleavage. Two highly UV-absorbing products of dR5P were amine derivatives of 5-methylene-2-pyrrolone and 2-formylpyrrole. The reaction of dR5P with certain amines generates a set of products that exhibit an interesting absorbance at 340nm and a high fluorescence. PMID:17850774

  9. Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidans.

    PubMed

    Gale, N L; Beck, J V

    1967-10-01

    The enzymes of the Calvin reductive pentose phosphate cycle and the hexose monophosphate pathway have been demonstrated in cell-free extracts of Thiobacillus ferrooxidans. This, together with analyses of the products of CO(2) fixation in cell-free systems, suggests that these pathways are operative in whole cells of this microorganism. Nevertheless, the amount of CO(2) fixed in these cell-free systems was limited by the type and amount of compound added as substrate. The inability of cell extracts to regenerate pentose phosphates and to perpetuate the cyclic fixation of CO(2) is partially attributable to low activity of triose phosphate dehydrogenase under the experimental conditions found to be optimal for the enzymes involved in the utilization of ribose-5-phosphate or ribulose-1,5-diphosphate as substrate for CO(2) incorporation. With the exception of ribulose-1,5-diphosphate, all substrates required the addition of adenosine triphosphate (ATP) or adenosine diphosphate (ADP) for CO(2) fixation. Under optimal conditions, with ribose-5-phosphate serving as substrate, each micromole of ATP added resulted in the fixation of 1.5 mumoles of CO(2), whereas each micromole of ADP resulted in 0.5 mumole of CO(2) fixed. These values reflect the activity of adenylate kinase in the extract preparations. The K(m) for ATP in the phosphoribulokinase reaction was 0.91 x 10(-3)m. Kinetic studies conducted with carboxydismutase showed K(m) values of 1.15 x 10(-4)m and 5 x 10(-2)m for ribulose-1,5-diphosphate and bicarbonate, respectively.

  10. Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

    PubMed Central

    Soh, Y; Song, B J; Jeng, J; Kallarakal, A T

    1998-01-01

    It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity. PMID:9657977

  11. A significant correlation between the plasma levels of coenzyme Q10 and vitamin B-6 and a reduced risk of coronary artery disease.

    PubMed

    Lee, Bor-Jen; Yen, Chi-Hua; Hsu, Hui-Chen; Lin, Jui-Yuan; Hsia, Simon; Lin, Ping-Ting

    2012-10-01

    Coronary artery disease (CAD) is the leading cause of death worldwide. The purpose of this study was to investigate the relationship between plasma levels of coenzyme Q10 and vitamin B-6 and the risk of CAD. Patients with at least 50% stenosis of one major coronary artery identified by cardiac catheterization were assigned to the case group (n = 45). The control group (n = 89) comprised healthy individuals with normal blood biochemistry. The plasma concentrations of coenzyme Q10 and vitamin B-6 (pyridoxal 5'-phosphate) and the lipid profiles of the participants were measured. Subjects with CAD had significantly lower plasma levels of coenzyme Q10 and vitamin B-6 compared to the control group. The plasma coenzyme Q10 concentration (β = 1.06, P = .02) and the ratio of coenzyme Q10 to total cholesterol (β = .28, P = .01) were positively correlated with vitamin B-6 status. Subjects with higher coenzyme Q10 concentration (≥516.0 nmol/L) had a significantly lower risk of CAD, even after adjusting for the risk factors for CAD. Subjects with higher pyridoxal 5'-phosphate concentration (≥59.7 nmol/L) also had a significantly lower risk of CAD, but the relationship lost its statistical significance after adjusting for the risk factors of CAD. There was a significant correlation between the plasma levels of coenzyme Q10 and vitamin B-6 and a reduced risk of CAD. Further study is needed to examine the benefits of administering coenzyme Q10 in combination with vitamin B-6 to CAD patients, especially those with low coenzyme Q10 level.

  12. Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

    PubMed

    Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

    2015-01-01

    A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucl