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Sample records for 5-phosphate reductoisomerase dxr

  1. The malarial drug target Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR): development of a 3-D model for identification of novel, structural and functional features and for inhibitor screening.

    PubMed

    Goble, Jessica L; Adendorff, Matthew R; de Beer, Tjaart A P; Stephens, Linda L; Blatch, Gregory L

    2010-01-01

    A three-dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure-checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non-apicomplexan DXR proteins. Furthermore, we have used the model to develop an efficient approach to screen for potential tool compounds for use in the rational design of novel DXR inhibitors.

  2. Catechol-rhodanine derivatives: Specific and promiscuous inhibitors of Escherichia coli deoxyxylulose phosphate reductoisomerase (DXR).

    PubMed

    Zinglé, Catherine; Tritsch, Denis; Grosdemange-Billiard, Catherine; Rohmer, Michel

    2014-07-15

    To develop more effective inhibitors than fosmidomycin, a natural compound which inhibits the deoxyxylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway, we designed molecules possessing on the one hand a catechol that is able to chelate the magnesium dication and on the other hand a group able to occupy the NADPH recognition site. Catechol-rhodanine derivatives (1-6) were synthesized and their potential inhibition was tested on the DXR of Escherichia coli. For the inhibitors 1 and 2, the presence of detergent in the enzymatic assays led to a dramatic decrease of the inhibition suggesting, that these compounds are rather promiscuous inhibitors. The compounds 4 and 5 kept their inhibition capacity in the presence of Triton X100 and could be considered as specific inhibitors of DXR. Compound 4 showed antimicrobial activity against Escherichia coli. The only partial protection of NADPH against the inhibition suggested that the catechol-rhodanine derivatives did not settle in the coenzyme binding site. This paper points out the necessity to include a detergent in the DXR enzymatic assays to avoid false positive when putative hydrophobic inhibitors are tested and especially when the IC50, are in the micromolar range.

  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.; 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. 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.

  5. 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.

  6. Antimicrobial mechanism of theaflavins: They target 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway

    PubMed Central

    Hui, Xian; Yue, Qiao; Zhang, Dan-Dan; Li, Heng; Yang, Shao-Qing; Gao, Wen-Yun

    2016-01-01

    1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 μM) and theaflavin-3,3′-digallate exhibiting the highest (IC50 14.9 μM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments. PMID:27941853

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2016-09-01

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

  9. Molecular cloning, characterization and expression analysis of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Aquilaria sinensis (Lour.) Gilg.

    PubMed

    Liu, Juan; Xu, Yanhong; Liang, Liang; Wei, Jianhe

    2015-06-01

    The major constituents of agarwood oils are sesquiterpenes that are obtained from isoprenoid precursors through the plastidial methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate pathway. In this study, a novel full-length cDNA of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), which was the second key enzyme in the plastid MEP pathway of sesquiterpenes biosynthesis was isolated from the stem of Aquilaria sinensis (Lour.) Gilg by the methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique for the first time, and named as AsDXR. The full-length cDNA of AsDXR was 1768 bp, containing a 1437 bp open reading frame (ORF) encoding a polypeptide of 478 amino acids with a molecular weight of 51.859 kD and the theoretical isoelectric point of 6.29. Comparative and bioinformatic analysis of the deduced AsDXR protein showed extensive homology with DXRs from other plant species, especially Theobroma cacao and Gossypium barbadense, and contained a conserved transit peptide for plastids, and extended pro-rich region and a highly conserved NADPH-binding motif owned by all plant DXRs. Southern blot analysis indicated that AsDXR belonged to a small gene family. Tissue expression pattern analysis revealed that AsDXR expressed strongly in root and stem, but weakly in leaf. Additionally, AsDXR expression was found to be activated by exogenous elicitor of MeJA (methyl jasmonate). The contents of three sesquiterpenes (α-guaiene, α-humulene and Δ-guaiene) were significantly induced by MeJA. This study enables us to further elucidate the role of AsDXR in the biosynthesis of agarwood sesquiterpenes in A. sinensis at the molecular level.

  10. 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

    2014-06-03

    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.

  11. Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

    PubMed

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Yu, Xiaohong; Hao, Xiaolong; Huang, Qiang; Xiao, Jianbo; Cui, Lijie; Kai, Guoyin

    2014-09-01

    Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g(-1) DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag(+) than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g(-1) DW by YE, Ag(+), and YE-Ag(+) treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.

  12. A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-D-xylulose 5-phosphate synthase.

    PubMed

    Sauret-Güeto, Susanna; Urós, Eva María; Ibáñez, Ester; Boronat, Albert; Rodríguez-Concepción, Manuel

    2006-02-06

    The 2-C-methyl-D-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.

  13. Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J.; Patel, Trishal; Bases, Jessica; Jose, Geraldine San; Jackson, Emily R.; Dowd, Cynthia S.; Couch, Robin D.

    2014-01-01

    The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. 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

  17. 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

  18. Functional identification and differential expression of 1-deoxy-D-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies).

    PubMed

    Phillips, Michael A; Walter, Michael H; Ralph, Steven G; Dabrowska, Paulina; Luck, Katrin; Urós, Eva Maria; Boland, Wilhelm; Strack, Dieter; Rodríguez-Concepción, Manuel; Bohlmann, Jörg; Gershenzon, Jonathan

    2007-10-01

    Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway

  19. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase

    PubMed Central

    Mahmoud, Soheil S.; Croteau, Rodney B.

    2001-01-01

    Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition. PMID:11427737

  20. Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes.

    PubMed

    Kim, Yeon-Bok; Kim, Sang-Min; Kang, Min-Kyoung; Kuzuyama, Tomohisa; Lee, Jong Kyu; Park, Seung-Chan; Shin, Sang-Chul; Kim, Soo-Un

    2009-05-01

    Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated

  1. Ketol-acid reductoisomerase enzymes and methods of use

    DOEpatents

    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.

  2. Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

    PubMed Central

    Kirby, James; Nishimoto, Minobu; Chow, Ruthie W. N.; Baidoo, Edward E. K.; Wang, George; Martin, Joel; Schackwitz, Wendy; Chan, Rossana; Fortman, Jeffrey L.

    2014-01-01

    Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP. PMID:25326299

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

    MedlinePlus

    ... 5'-phosphate-dependent epilepsy pyridoxal 5'-phosphate-dependent epilepsy Enable Javascript to view the expand/collapse boxes. ... All Close All Description Pyridoxal 5'-phosphate-dependent epilepsy is a condition that involves seizures beginning soon ...

  4. Biomedical aspects of pyridoxal 5'-phosphate availability.

    PubMed

    di Salvo, Martino L; Safo, Martin K; Contestabile, Roberto

    2012-01-01

    The biologically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor in over 160 enzyme activities involved in a number of metabolic pathways, including neurotransmitter synthesis and degradation. In humans, PLP is recycled from food and from degraded PLP-dependent enzymes in a salvage pathway requiring the action of pyridoxal kinase, pyridoxine 5'-phosphate oxidase and phosphatases. Once pyridoxal 5'-phosphate is made, it is targeted to the dozens different apoenzymes that need it as a cofactor. The regulation of the salvage pathway and the mechanism of addition of PLP to the apoenzymes are poorly understood and represent a very challenging research field. Severe neurological disorders, such as convulsions and epileptic encephalopathy, result from a reduced availability of pyridoxal 5'-phosphate in the cell, due to inborn errors in the enzymes of the salvage pathway or other metabolisms and to interactions of drugs with PLP or pyridoxal kinase. Multifactorial neurological pathologies, such as autism, schizophrenia, Alzheimer's disease, Parkinson's disease and epilepsy have also been correlated to inadequate intracellular levels of PLP.

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Riboflavin-5-phosphate. 582.5697 Section 582.5697 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  6. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Riboflavin-5-phosphate. 582.5697 Section 582.5697 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  7. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Riboflavin-5-phosphate. 582.5697 Section 582.5697 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... 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, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Riboflavin-5-phosphate. 582.5697 Section 582.5697 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... 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, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Riboflavin-5-phosphate. 582.5697 Section 582.5697 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  10. 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…

  11. 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.

  12. 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.

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

    SciTech Connect

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

    1986-02-15

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

  14. Combined activity of interleukin-1 alpha or TNF-alpha and doxorubicin on multidrug resistant cell lines: evidence that TNF and DXR have synergistic antitumor and differentiation-inducing effects.

    PubMed

    Borsellino, N; Crescimanno, M; Flandina, C; Flugy, A; D'Alessandro, N

    1994-01-01

    We report on the antiproliferative effects that interleukin-1 alpha (IL-1) or TNF-alpha (TNF) in combination with doxorubicin (DXR) exert on DXR-sensitive (B16 melanoma, Friend, K562 and CCRF/CEM leukemias) and -resistant (B16-DXR, FLC-DXR, K562-DXR) cell lines in vitro. Multidrug resistance (MDR) of the latter lines entails cross-resistance to vincristine and overexpression of P-glycoprotein. Il-1 showed only a very marginal growth inhibitory activity and the effects of its combination with DXR were essentially additive in all the cell lines, except in chemosensitive B16, where a slight synergism occurred. TNF demonstrated greater antiproliferative activity in the MDR B16 and Friend tumors than in their parent variants. The combination of TNF and DXR produced synergistic growth inhibition in B16, K562 and, particularly, also in the MDR sublines of these two tumors. In addition, TNF and DXR induced synergistically erythroid differentiation in K562 and multidirectional differentiation in K562-DXR. The synergism was critically schedule-dependent in that it was achieved only when DXR application preceded or was simultaneous with that of TNF. Finally, TNF did not modify drug accumulation and retention in the cells. Our present findings stress especially the fact that DXR and TNF may exert useful antitumor synergism even in MDR lines; however, it is not likely that their interaction will occur at the specific MDR process level.

  15. 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

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

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    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

  18. Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad.

    PubMed

    Ramak, Parvin; Kazempour Osaloo, Shahrokh; Ebrahimzadeh, Hassan; Sharifi, Mozafar; Behmanesh, Mehrdad

    2013-09-01

    Carvacrol is a major component of Satureja khuzistanica Jamzad (≤90%) that has significant antimicrobial and antioxidant properties. Considering the specific capabilities of S. khuzistanica to produce highly pure carvacrol, this plant is an important potential source of carvacrol that could address the abundant consumption and increasing demand for this monoterpene in current world markets. This research was performed to better understand the process of biosynthesis and accumulation of carvacrol in S. khuzistanica. Tests were performed on shoot cultures of S. khuzistanica in Linsmaier-Skoog (LS) medium treated with different concentrations of fosmidomycin (an inhibitor of the non-mevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway) for 21 days at the following concentrations: 0, 10, 25, 50, 75 and 100 μM. The present study demonstrated that the MEP pathway is the major pathway that provides IPP for the biosynthesis of carvacrol, and the expression and activity levels of the DXR enzyme have a critical effect on carvacrol biosynthesis. Surprisingly, Mevinolin at concentrations of 75 and 100 μM increased the carvacrol content and the DXR activity and gene expression in S. khuzistanica plantlets.

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

    SciTech Connect

    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.

  20. Molecular epidemiology of malaria in Cameroon. XXV. In vitro activity of fosmidomycin and its derivatives against fresh clinical isolates of Plasmodium falciparum and sequence analysis of 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

    PubMed

    Tahar, Rachida; Basco, Leonardo K

    2007-08-01

    The in vitro activities of fosmidomycin derivatives, chloroquine, and pyrimethamine were assessed by the radioisotopic assay in clinical isolates of Plasmodium falciparum. In a series of experiments with RPMI 1640 medium-10% fetal bovine serum, the geometric mean 50% inhibitory concentrations (IC(50)s) (n = 34) for fosmidomycin and FR900098 were 301 nM and 118 nM, respectively. In another series of experiments, the geometric mean IC(50)s (n = 33) for fosmidomycin and TH II46 were 413 nM and 249 nM, respectively. The IC(50)s were 2-3 times lower with RPMI-10% fetal bovine serum than the IC(50)s obtained with RPMI-10% human serum. FR900098 and TH II46 were 2.6 and 1.7 times more potent, respectively, than fosmidomycin. There was no correlation between chloroquine or pyrimethamine and fosmidomycin, which suggested the absence of in vitro cross-resistance. Sequence analysis showed five amino acid substitutions, but their possible relationship with the response to fosmidomycin is not clear. Fosmidomycin derivatives are promising candidates for further development.

  1. Purification and characterization of pyridoxine 5'-phosphate phosphatase from Sinorhizobium meliloti.

    PubMed

    Tazoe, Masaaki; Ichikawa, Keiko; Hoshino, Tatsuo

    2005-12-01

    Here we report the purification and biochemical characterization of a pyridoxine 5'-phosphate phosphatase involved in the biosynthesis of pyridoxine in Sinorhizobium meliloti. The phosphatase was localized in the cytoplasm and purified to electrophoretic homogeneity by a combination of EDTA/lysozyme treatment and five chromatography steps. Gel-filtration chromatography with Sephacryl S-200 and SDS/PAGE demonstrated that the protein was a monomer with a molecular size of approximately 29 kDa. The protein required divalent metal ions for pyridoxine 5'-phosphate phosphatase activity, and specifically catalyzed the removal of Pi from pyridoxine and pyridoxal 5'-phosphates at physiological pH (about 7.5). It was inactive on pyridoxamine 5'-phosphate and other physiologically important phosphorylated compounds. The enzyme had the same Michaelis constant (K(m)) of 385 muM for pyridoxine and pyridoxal 5'-phosphates, but its specific constant [maximum velocity (V(max))/K(m)] was nearly 2.5 times higher for the former than for the latter.

  2. Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

    PubMed

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

    2016-01-01

    Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

  3. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

    PubMed

    Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

    2012-12-01

    The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

  4. Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

    PubMed Central

    2011-01-01

    Background Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. Results Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. Conclusion The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether. PMID:21995815

  5. 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

  6. Role of the pyridine nitrogen in pyridoxal 5'-phosphate catalysis: activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5'-phosphate.

    PubMed

    Griswold, Wait R; Toney, Michael D

    2011-09-21

    Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.

  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

    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.

  9. 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

  10. Pyridoxal 5'-phosphate binding in lysine-modified PAMAM dendrimers: a biomimetic approach.

    PubMed

    Hsien, Kuang-Chan; Chen, Hui-Ting; Chen, Yi-Chen; Chen, Yeh-Long; Lu, Chi-Yu; Kao, Chai-Lin

    2009-08-20

    (G:3-7)-dendri-PAMAM-(APO-Phe-Lys)(x) (2, APO = aminopropanol, Phe = phenylalanine, Lys = lysine) were prepared and used in a binding study with pyridoxal 5'-phosphate. The results revealed a positive dendritic effect, and binding ability was found to vary with the environment. (G:3-7)-dendri-PAMAM-(APO-Phe-Lys)(x) (2) demonstrated better binding ability at higher pH, and protonation of lysine was considered to affect binding. The strongest binding affinity was K(b) = 254.3 mM(-1) at pH 9, which was shown by (G:7)-dendri-PAMAM-(APO-Phe-Lys)(490) (2e).

  11. 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.

  12. 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.

  13. 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

  14. Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis.

    PubMed

    Okada, Kazunori; Kawaide, Hiroshi; Kuzuyama, Tomohisa; Seto, Haruo; Curtis, Ian S; Kamiya, Yuji

    2002-06-01

    Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2- C-methyl- D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of ent-kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of ent-kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that ent-kaurene is mainly produced through the nonmevalonate pathway in the plastid.

  15. 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.

  16. Molecular characterization of novel pyridoxal-5'-phosphate-dependent enzymes from the human microbiome.

    PubMed

    Fleischman, Nicholas M; Das, Debanu; Kumar, Abhinav; Xu, Qingping; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Elsliger, Marc-André; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Wilson, Ian A; Toney, Michael D

    2014-08-01

    Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.

  17. Structure and catalytic mechanism of the cytosolic D-ribulose-5-phosphate 3-epimerase from rice.

    PubMed

    Jelakovic, Stefan; Kopriva, Stanislav; Süss, Karl Heinz; Schulz, Georg E

    2003-02-07

    Cytosolic D-ribulose-5-phosphate 3-epimerase from rice was crystallized after EDTA treatment and structurally elucidated by X-ray diffraction to 1.9A resolution. A prominent Zn(2+) site at the active center was established in a soaking experiment. The structure was compared with that of the EDTA-treated crystalline enzyme from the chloroplasts of potato plant leaves showing some structural differences, in particular the "closed" state of a strongly conserved mobile loop covering the substrate at its putative binding site. The previous proposal for the active center was confirmed and the most likely substrate binding position and conformation was derived from the locations of the bound zinc and sulfate ions and of three water molecules. Assuming that the bound zinc ion is an integral part of the enzyme, a reaction mechanism involving a well-stabilized cis-enediolate intermediate is suggested.

  18. Selection of a new whole cell biocatalyst for the synthesis of 2-deoxyribose 5-phosphate.

    PubMed

    Valino, Ana L; Palazzolo, Martín A; Iribarren, Adolfo M; Lewkowicz, Elizabeth

    2012-01-01

    2-deoxyribose 5-phosphate (DR5P) is a key intermediate in the biocatalyzed preparation of deoxyribonucleosides. Therefore, DR5P production by means of simpler, cleaner, and economic pathways becomes highly interesting. One strategy involves the use of bacterial whole cells containing DR5P aldolase as biocatalyst for the aldol addition between acetaldehyde and D: -glyceraldehyde 3-phosphate or glycolytic intermediates that in situ generate the acceptor substrate. In this work, diverse microorganisms capable of synthesizing DR5P were selected by screening several bacteria genera. In particular, Erwinia carotovora ATCC 33260 was identified as a new biocatalyst that afforded 14.1-mM DR5P starting from a cheap raw material like glucose.

  19. 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

    2013-06-07

    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.

  20. 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

  1. Inhibitory effect of pyridoxal 5'-phosphate on the DNA binding site of ATP-dependent deoxyribonuclease from Bacillus laterosporus.

    PubMed

    Fujiyoshi, T; Nakayama, J; Anai, M

    1981-04-01

    Bacillus laterosporus ATP-dependent deoxyribonuclease has been found to be inhibited by pyridoxal 5'-phosphate. The inhibition is specific for pyridoxal 5'-phosphate and pyridoxal which are required in relatively high concentrations. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine are ineffective. The inhibition is reversed by dilution or dialysis but can be changed to an irreversible inactivation by reduction of the enzyme . pyridoxal 5'-phosphate complex with sodium borohydride. The compound is a competitive inhibitor with respect to DNA but not ATP. Moreover, the presence of DNA substrate protects the enzyme against this inactivation but the presence of ATP shows no effect. The reduced enzyme . pyridoxal 5'-phosphate complex displays a new absorption maximum at 325 nm and a fluorescence emission at 390-400 nm when excited at 325 nm which are characteristic for epsilon-N-(phosphopyridoxyl)lysine. Thus, B. laterosporus DNase appears to have an essential lysine residue at the DNA binding site of the enzyme, and the enzyme possess two different active sites, a DNA binding site and an ATP binding site.

  2. Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme.

    PubMed

    Balasundaram, D; Tyagi, A K

    1989-08-01

    Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.

  3. L-Arabinose-sensitive, L-ribulose 5-phosphate 4-epimerase-deficient mutants of Escherichia coli.

    PubMed

    ENGLESBERG, E; ANDERSON, R L; WEINBERG, R; LEE, N; HOFFEE, P; HUTTENHAUER, G; BOYER, H

    1962-07-01

    Englesberg, E. (University of Pittsburgh, Pittsburgh, Pa.), R L. Anderson, R. Weinberg, N. Lee, P. Hoffee, G. Huttenhauer, and H. Boyer. l-Arabinose-sensitive, l-ribulose 5-phosphate 4-epimerase-deficient mutants of Escherichia coli. J. Bacteriol. 84:137-146. 1962-l-Arabinose-negative mutants of Escherichia coli B/r, ara-53 and ara-139, are deficient in the enzyme l-ribulose 5-phosphate 4-epimerase; ara-53, further analyzed, accumulates large quantities of l-ribulose 5-phosphate when incubated with l-arabinose. The mutant sites are closely linked to the left of the previously ordered l-arabinose mutant sites, and probably represent the structural gene for l-ribulose 5-phosphate 4-epimerase (gene D) in the l-arabinose operon. The inducible levels of l-arabinose isomerase and l-ribulose 5-phosphate 4-epimerase vary correspondingly as a result of mutation in the structural gene for l-ribulokinase (gene B), further substantiating the dual structural and regulatory function of this gene locus. Ara-53 and ara-139 are strongly inhibited by l-arabinose and give rise to l-arabinose-resistant mutants. The one resistant mutant analyzed still lacks the 4-epimerase but is deficient in l-ribulokinase and has increased l-arabinose isomerase activity, a characteristic of a type of mutation in the B gene. It is proposed that accumulation of l-ribulose 5-phosphate is responsible for the inhibition, and that mutation to resistance will involve mutation in the A, B, C, permease, or repressor genes, thus providing a direct method for isolating these types of l-arabinose-negative mutants. Glucose prevents and cures the l-arabinose inhibition.

  4. Formation of novel nucleosides from free base and sugar phosphate: aqueous reaction of 2-aminopyrimidine and ribose-5-phosphate.

    PubMed

    Mace, D C

    1983-11-30

    The facile formation of glycosylamines suggests that a base liberated by depurination might react at the free C1 position of the sugar phosphate from which it had been hydrolyzed, effectively repurinating the site. Model experiments testing this hypothesis demonstrate that such a reaction does take place. The primary product of a reaction between 2-aminopyrimidine (a model for guanine) and ribose-5-phosphate is characterized by enzymatic and chemical degradation, and UV spectra. It is shown to be a novel nucleoside with the base attached via its exocyclic amino group to the C1 of the ribose-5-phosphate.

  5. 6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

    PubMed

    Le, Simone Balzer; Heggeset, Tonje Marita Bjerkan; Haugen, Tone; Nærdal, Ingemar; Brautaset, Trygve

    2017-02-17

    D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg(2+) and Mn(2+) and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg(2+) and Mn(2+), and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.

  6. Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

    PubMed

    Huang, ShuoHao; Han, CaiYun; Ma, ZhenQiao; Zhou, Jie; Zhang, JianYun; Huang, LongQuan

    2017-03-01

    Vitamin B6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B6 vitamers were detected as compared with the control.

  7. Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.

    PubMed

    Vermeersch, Jacqueline J; Christmann-Franck, Serge; Karabashyan, Leon V; Fermandjian, Serge; Mirambeau, Gilles; Der Garabedian, P Arsène

    2004-01-01

    The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the epsilon-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K(i) = 40 microM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532-PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.

  8. 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.

  9. Critical hydrogen bonds and protonation states of pyridoxal 5'-phosphate revealed by NMR.

    PubMed

    Limbach, Hans-Heinrich; Chan-Huot, Monique; Sharif, Shasad; Tolstoy, Peter M; Shenderovich, Ilya G; Denisov, Gleb S; Toney, Michael D

    2011-11-01

    In this contribution we review recent NMR studies of protonation and hydrogen bond states of pyridoxal 5'-phosphate (PLP) and PLP model Schiff bases in different environments, starting from aqueous solution, the organic solid state to polar organic solution and finally to enzyme environments. We have established hydrogen bond correlations that allow one to estimate hydrogen bond geometries from (15)N chemical shifts. It is shown that protonation of the pyridine ring of PLP in aspartate aminotransferase (AspAT) is achieved by (i) an intermolecular OHN hydrogen bond with an aspartate residue, assisted by the imidazole group of a histidine side chain and (ii) a local polarity as found for related model systems in a polar organic solvent exhibiting a dielectric constant of about 30. Model studies indicate that protonation of the pyridine ring of PLP leads to a dominance of the ketoenamine form, where the intramolecular OHN hydrogen bond of PLP exhibits a zwitterionic state. Thus, the PLP moiety in AspAT carries a net positive charge considered as a pre-requisite to initiate the enzyme reaction. However, it is shown that the ketoenamine form dominates in the absence of ring protonation when PLP is solvated by polar groups such as water. Finally, the differences between acid-base interactions in aqueous solution and in the interior of proteins are discussed. This article is part of a special issue entitled: Pyridoxal Phosphate Enzymology.

  10. Changes in plasma pyridoxal 5'-phosphate concentration during pregnancy stages in Japanese women.

    PubMed

    Shibata, Katsumi; Tachiki, Akiko; Mukaeda, Kana; Fukuwatari, Tsutomu; Sasaki, Satoshi; Jinno, Yoshiki

    2013-01-01

    Most Japanese women do not consume the estimated average requirement of vitamin B6 (1.7 mg/d) during pregnancy. Nevertheless, these deficiencies are not reported. We investigated a nutritional biomarker of vitamin B6 in pregnant Japanese women as well as their vitamin B6 intakes. Vitamin B6 intakes in the first, second, and third trimesters of pregnancy, and 1 mo after delivery were 0.79±0.61 (n=56), 0.81±0.29 (n=71), 0.90±0.35 (n=92), and 1.00±0.31 (n=44) mg/d, respectively. Plasma pyridoxal 5'-phosphate (PLP) concentrations in the first, second, and third trimesters of pregnancy, and 1 mo after delivery were 57.1±27.6 (n=56), 23.3±16.7 (n=71), 18.3±12.5 (n=92), and 43.9±33.4 (n=44) nmol/L, respectively. The plasma concentrations significantly decreased in the second and third trimesters of pregnancy compared to values from the first trimester (p<0.05), and these concentrations returned to the values of the first trimester of pregnancy 1 mo after birth.

  11. 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

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

    PubMed

    Singh, Vivek Kumar; Ghosh, Indira

    2013-09-02

    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.

  13. Prevention of Proliferative Vitreoretinopathy by Suppression of Phosphatidylinositol 5-Phosphate 4-Kinases

    PubMed Central

    Ma, Gaoen; Duan, Yajian; Huang, Xionggao; Qian, Cynthia X.; Chee, Yewlin; Mukai, Shizuo; Cui, Jing; Samad, Arif; Matsubara, Joanne Aiko; Kazlauskas, Andrius; D'Amore, Patricia A.; Gu, Shuyan; Lei, Hetian

    2016-01-01

    Purpose Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -β) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -β and whether suppression of PI5P4Kα and -β would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). Methods PI5P4Kα and -β encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -β by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -β knocked-down ARPE-19 cells. Expression of PI5P4Kα and -β was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. Results We found that vitreous enhanced expression of PI5P4Kα and -β in RPE cells and that knocking down PI5P4Kα and -β abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -β in the human PI5P4Kα and -β knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -β abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -β was abundant in epiretinal membranes from PVR grade C patients. Conclusions The findings from this study indicate that PI5P4Kα and -β could be novel therapeutic targets for the treatment of PVR. PMID:27472081

  14. 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.

  15. 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.

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

    PubMed

    Feng, Zaiping; Mu, Wanmeng; Jiang, Bo

    2013-05-01

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

  17. 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.

  18. Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli.

    PubMed

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

    2006-06-01

    2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

  19. 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.

  20. Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates.

    PubMed

    Cheng, Feng; Oldfield, Eric

    2004-10-07

    We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock. The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically. The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008). The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues. Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme. The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH. Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E. coli enzyme). Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes.

  1. Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis

    PubMed Central

    Meile, Leo; Rohr, Lukas M.; Geissmann, Thomas A.; Herensperger, Monique; Teuber, Michael

    2001-01-01

    A d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probiotic Bifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with d-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme. Km values for d-xylulose 5-phosphate and d-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (named xfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta and guaA, were localized adjacent to xfp on the B. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes in Mycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase. PMID:11292814

  2. 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

  3. 1-Deoxy-d-Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

    PubMed Central

    Hahn, Frederick M.; Eubanks, Lisa M.; Testa, Charles A.; Blagg, Brian S. J.; Baker, Jonathan A.; Poulter, C. Dale

    2001-01-01

    In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and d-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-d-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-d-xylulose 5-phosphate synthase A and B, containing a His6 tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-d-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-d-xylulose 5-phosphate synthase A and B: Kmpyruvate = 0.61 and 3.0 mM, Kmd-glyceraldehyde 3-phosphate = 150 and 120 μM, and Vmax = 1.9 and 1.4 μmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-d-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11. PMID:11114895

  4. NMR studies of coupled low- and high-barrier hydrogen bonds in pyridoxal-5'-phosphate model systems in polar solution.

    PubMed

    Sharif, Shasad; Denisov, Gleb S; Toney, Michael D; Limbach, Hans-Heinrich

    2007-05-16

    The 1H and 15N NMR spectra of several 15N-labeled pyridoxal-5'-phosphate model systems have been measured at low temperature in various aprotic and protic solvents of different polarity, i.e., dichloromethane-d2, acetonitrile-d3, tetrahydrofuran-d8, freon mixture CDF3/CDClF2, and methanol. In particular, the 15N-labeled 5'-triisopropyl-silyl ether of N-(pyridoxylidene)-tolylamine (1a), N-(pyridoxylidene)-methylamine (2a), and the Schiff base with 15N-2-methylaspartic acid (3a) and their complexes with proton donors such as triphenylmethanol, phenol, and carboxylic acids of increasing strength were studied. With the use of hydrogen bond correlation techniques, the 1H/15N chemical shift and scalar coupling data could be associated with the geometries of the intermolecular O1H1N1 (pyridine nitrogen) and the intramolecular O2H2N2 (Schiff base) hydrogen bonds. Whereas O1H1N1 is characterized by a series of asymmetric low-barrier hydrogen bonds, the proton in O2H2N2 faces a barrier for proton transfer of medium height. When the substituent on the Schiff base nitrogen is an aromatic ring, the shift of the proton in O1H1N1 from oxygen to nitrogen has little effect on the position of the proton in the O2H2N2 hydrogen bond. By contrast, when the substituent on the Schiff base nitrogen is a methyl group, a proton shift from O to N in O1H1N1 drives the tautomeric equilibrium in O2H2N2 from the neutral O2-H2...N2 to the zwitterionic O2-...H2-N(2+) form. This coupling is lost in aqueous solution where the intramolecular O2H2N2 hydrogen bond is broken by solute-solvent interactions. However, in methanol, which mimics hydrogen bonds to the Schiff base in the enzyme active site, the coupling is preserved. Therefore, the reactivity of Schiff base intermediates in pyridoxal-5'-phosphate enzymes can likely be tuned to the requirements of the reaction being catalyzed by differential protonation of the pyridine nitrogen.

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

    PubMed

    Guggisberg, Ann M; Park, Jooyoung; Edwards, Rachel L; Kelly, Megan L; Hodge, Dana M; Tolia, Niraj H; Odom, Audrey R

    2014-07-24

    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. Here 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 homologues in plants and bacteria, other HAD proteins may be MEP pathway regulators.

  6. Pyridoxal 5'-phosphate mediated preparation of immobilized metal affinity material for highly selective and sensitive enrichment of phosphopeptides.

    PubMed

    Wang, Qian; He, Xiao-Mei; Chen, Xi; Zhu, Gang-Tian; Wang, Ren-Qi; Feng, Yu-Qi

    2017-04-01

    Phosphorylation is a crucial post-translational modification, which plays pivotal roles in various biological processes. Analysis of phosphopeptides by mass spectrometry (MS) is intractable on account of their low stoichiometry and the ion suppression from non-phosphopeptides. Thus, enrichment of phosphopeptides before MS analysis is indispensable. In this work, we employed pyridoxal 5'-phosphate (PLP), as an immobilized metal affinity chromatography (IMAC) ligand for the enrichment of phosphopeptides. PLP was grafted onto several substrates such as silica (SiO2), oxidized carbon nanotube (OCNT) and silica coated magnetic nanoparticles (Fe3O4@SiO2). Then the metal ions Fe(3+), Ga(3+) and Ti(4+) were incorporated for the selective enrichment of phosphopeptides. It is indicated that Fe3O4@SiO2-PLP-Ti(4+) has a superior selectivity towards phosphopeptides under as much as 1000-fold interferences of non-phosphopeptides. Further, Fe3O4@SiO2-PLP-Ti(4+) exhibited high efficiency in selective enrichments of phosphopeptides from complex biological samples, including human serum and tryptic digested non-fat milk. Finally, Fe3O4@SiO2-PLP-Ti(4+) was successfully employed in the sample pretreatment for profiling phosphopeptides in a tryptic digest of rat brain proteins. Our experimental results evidenced a great potential of this new chelator-based material in phosphoproteomics study.

  7. Characterization of a pyridoxal-5'-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Takahashi, Narumi; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2014-11-01

    A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44 mM and 0.84 mM, respectively. The molecular mass was estimated to be 150 kDa, which was composed of two identical subunits with molecular mass of 76.5 kDa. The enzyme contained one mol of pyridoxal 5'-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.

  8. 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.

  9. 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.

  10. The effect of temperature on ribose-5-phosphate isomerase from a mesophile, Thiobacillus thioparus, and a thermophile, Bacillus caldolyticus.

    PubMed

    Middaugh, C R; MacElroy, R D

    1976-06-01

    The enzyme ribose-5-phosphate isomerase [EC 5.3.1.6] was partially purified from a mesophilic organism, Thiobacillus thioparus, and from an extreme thermophile, Bacillus caldolyticus. The stability and kinetics of the two enzymes were compared with regard to temperature in the presence of a series of neutral salts and alcohols. The thermal stability of both enzymes was altered such that the salts (NH4)2SO4, NaCl, KCl, and LiCl increased stability, while LiBr, CaCl2, methanol, ethanol, and 1-propanol decreased stability. Ethylene glycol had little effect on the mesophilic enzyme, but increased the stability of the thermophilic protein. The kinetics of both enzymes were also affected by the salts and alcohols, and Arrhenius plots of two kinetic parameters, Km and Vmax, displayed discontinuities, or sharp changes in slope, at characteristic temperatures, TD. Neutral salts and alcohols altered the temperature of discontinuity in a sequence similar to that observed in studies of thermal stability. It is suggested that the slope change is due to temperature-dependent alterations in the enzymes at specific, but undefined, loci at the active site, although no evidence is offered for the absence of a larger conformation change in the entire enzyme.

  11. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    PubMed

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.

  12. Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution

    PubMed Central

    Sagong, Hye-Young; Kim, Kyung-Jin

    2017-01-01

    Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity. PMID:28095457

  13. 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.

  14. Short communication: empirical and mechanistic evidence for the role of pyridoxal-5'-phosphate in the generation of methanethiol from methionine.

    PubMed

    Wolle, D D; Banavara, D S; Rankin, S A

    2006-12-01

    The catabolism of the sulfur-containing AA Met to form flavor-active volatile sulfur compounds (VSC) is an important mechanism in flavor development during cheese maturation. Numerous enzymes catalyzing AA catabolism require the presence of the cofactor pyri-doxal-5'-phosphate (PLP). In fact, reports have shown that some of these reactions can be catalyzed by PLP alone, albeit at a reduced rate. We hypothesized that, as a specific application in cheese flavor reactions, PLP can react directly with free Met to generate a specific VSC, methanethiol (MTH). In this study, the ability of PLP to catalyze MTH generation from Met was examined under "cheeselike" conditions of salt and pH. Methionine and varying concentrations of PLP were incubated in a buffer (pH 5.2 + 4.0% NaCl) analogous to the aqueous phase of aged Cheddar cheese. Samples were analyzed using headspace solid-phase microextraction, and relative concentrations of VSC were determined by gas chromatography-mass spectrometry. Results showed MTH, dimethyl disulfide, and dimethyl trisulfide production when Met and PLP were incubated together at 7 degrees C (cheese-aging temperature). These results indicate that the production of VSC from Met can occur nonenzymatically as catalyzed by free PLP.

  15. Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: a single-tryptophan protein.

    PubMed

    Kaur, Preet Kamal; Supin, Jakka S; Rashmi, S; Singh, Sushma

    2014-08-01

    Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.

  16. Coupling of functional hydrogen bonds in pyridoxal-5'-phosphate-enzyme model systems observed by solid-state NMR spectroscopy.

    PubMed

    Sharif, Shasad; Schagen, David; Toney, Michael D; Limbach, Hans-Heinrich

    2007-04-11

    We present a novel series of hydrogen-bonded, polycrystalline 1:1 complexes of Schiff base models of the cofactor pyridoxal-5'-phosphate (PLP) with carboxylic acids that mimic the cofactor in a variety of enzyme active sites. These systems contain an intramolecular OHN hydrogen bond characterized by a fast proton tautomerism as well as a strong intermolecular OHN hydrogen bond between the pyridine ring of the cofactor and the carboxylic acid. In particular, the aldenamine and aldimine Schiff bases N-(pyridoxylidene)tolylamine and N-(pyridoxylidene)methylamine, as well as their adducts, were synthesized and studied using 15N CP and 1H NMR techniques under static and/or MAS conditions. The geometries of the hydrogen bonds were obtained from X-ray structures, 1H and 15N chemical shift correlations, secondary H/D isotope effects on the 15N chemical shifts, or directly by measuring the dipolar 2H-15N couplings of static samples of the deuterated compounds. An interesting coupling of the two "functional" OHN hydrogen bonds was observed. When the Schiff base nitrogen atoms of the adducts carry an aliphatic substituent such as in the internal and external aldimines of PLP in the enzymatic environment, protonation of the ring nitrogen shifts the proton in the intramolecular OHN hydrogen bond from the oxygen to the Schiff base nitrogen. This effect, which increases the positive charge on the nitrogen atom, has been discussed as a prerequisite for cofactor activity. This coupled proton transfer does not occur if the Schiff base nitrogen atom carries an aromatic substituent.

  17. The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

    PubMed

    Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

    2014-02-01

    The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed.

  18. 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.

  19. 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.

  20. 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.

  1. The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate.

    PubMed

    Remani, Sreevidya; Sun, Jiakang; Kotaria, Rusudan; Mayor, June A; Brownlee, June M; Harrison, David H T; Walters, D Eric; Kaplan, Ronald S

    2008-12-01

    The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH(2)CH(2)NH(3) (+)) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine

  2. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

    PubMed

    Gonzalez, Soledad Natalia; Valsecchi, Wanda Mariela; Maugeri, Dante; Delfino, José María; Cazzulo, Juan José

    2017-01-01

    The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these

  3. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi

    PubMed Central

    Gonzalez, Soledad Natalia; Valsecchi, Wanda Mariela; Maugeri, Dante; Delfino, José María; Cazzulo, Juan José

    2017-01-01

    The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these

  4. [Roles of reactive oxygen species in Streptomyces pactum Act12-induced tanshinone production in Salvia miltiorrhiza hairy roots].

    PubMed

    Yan, Yan; Zhao, Xin; Zhang, Shun-Cang; Liu, Yan; Liang, Zong-Suo

    2014-06-01

    Our previous research indicated that the Streptomyces pactum Act12 (Act12) had a certain promotional effect on tanshinone accumulation and up-regulated the expression of genes 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in Salvia miltiorrhiza hairy roots. This study focuses on the roles of reactive oxygen species in S. pactum Act12-induced tanshinone production in S. miltiorrhiza hairy roots. The 4% Act12, 4% Act12 + CAT and 4% Act12 + SOD were added to S. miltiorrhiza hairy root and subcultured for 21 days, the dry weight, contents of reactive oxygen species, contents of tanshinones and expression of HMGR and DXR were determined at different harvest-time. The generation of reactive oxygen species (ROS) in S. miltiorrhiza hairy roots was triggered by 4% Act12 treatment. The relative expressions of genes HMGR and DXR in 4% Act12 treatment were 32.4 and 4.8-fold higher than those in the control. And the total tanshinone in the hairy roots was 10.2 times higher than that of the control. The CAT and SOD could significantly inhibit the ROS accumulation and relative expressions of genes HMGR and DXR in 4% Act12 treatment, which induced the total tanshinone content was decreased by 74.6% comparing with the 4% Act12 treatment. ROS mediated Act12-induced tanshinone production. The Act12 may be via the ROS signal channel to activate the tanshinone biosynthesis pathways. Thereby the tanshinon content in hairy roots was increased.

  5. 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

  6. Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway.

    PubMed

    Perez-Gil, Jordi; Uros, Eva Maria; Sauret-Güeto, Susanna; Lois, L Maria; Kirby, James; Nishimoto, Minobu; Baidoo, Edward E K; Keasling, Jay D; Boronat, Albert; Rodriguez-Concepcion, Manuel

    2012-01-01

    A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

  7. 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.

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

    SciTech Connect

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

    1998-09-01

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

  9. 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.

  10. 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.

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

    SciTech Connect

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

    2006-01-01

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

  12. Synthesis of hydrolysis-resistant pyridoxal 5'-phosphate analogs and their biochemical and X-ray crystallographic characterization with the pyridoxal phosphatase chronophin.

    PubMed

    Knobloch, Gunnar; Jabari, Nauras; Stadlbauer, Sven; Schindelin, Hermann; Köhn, Maja; Gohla, Antje

    2015-06-15

    A set of phosphonic acid derivatives (1-4) of pyridoxal 5'-phosphate (PLP) was synthesized and characterized biochemically using purified murine pyridoxal phosphatase (PDXP), also known as chronophin. The most promising compound 1 displayed primarily competitive PDXP inhibitory activity with an IC50 value of 79μM, which was in the range of the Km of the physiological substrate PLP. We also report the X-ray crystal structure of PDXP bound to compound 3, which we solved to 2.75Å resolution (PDB code 5AES). The co-crystal structure proves that compound 3 binds in the same orientation as PLP, and confirms the mode of inhibition to be competitive. Thus, we identify compound 1 as a PDXP phosphatase inhibitor. Our results suggest a strategy to design new, potent and selective PDXP inhibitors, which may be useful to increase the sensitivity of tumor cells to treatment with cytotoxic agents.

  13. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway.

    PubMed

    Zhao, Yaru; Yang, Jianming; Qin, Bo; Li, Yonghao; Sun, Yuanzhang; Su, Sizheng; Xian, Mo

    2011-06-01

    Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-D: -xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.

  14. Enzymatic production and emission of floral scent volatiles in Jasminum sambac.

    PubMed

    Bera, Paramita; Mukherjee, Chiranjit; Mitra, Adinpunya

    2017-03-01

    Floral scent composed of low molecular weight volatile organic compounds. The sweet fragrance of any evening blooming flower is dominated by benzenoid and terpenoid volatile compounds. Floral scent of Jasminum sambac (Oleaceae) includes three major benzenoid esters - benzylacetate, methylbenzoate, and methylsalicylate and three major terpene compounds viz. (E)-β-ocimene, linalool and α-farnesene. We analyzed concentrations and emission rates of benzenoids and terpenoids during the developmental stages of J. sambac flower. In addition to spatial emission from different floral parts, we studied the time-course mRNA accumulations of phenylalanine ammonia-lyase (PAL) and the two representative genes of terpenoid pathway, namely 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and terpene synthase (TPS). Further, in vitro activities of several enzymes of phenylpropanoid/benzenoid pathway viz., PAL and acetyl-coenzyme A: benzylalcohol acetyltransferase (BEAT), S-adenosyl-l-methionine: benzoic acid carboxyl methyl transferase (BAMT) and S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase (SAMT) were studied. All the above enzyme activities along with the in vitro activities of DXR and TPS were found to follow a certain rhythm as observed in the emission of different benzenoid and terpenoid compounds. Linalool emission peaked after petal opening and coincided with maximal expression of JsTPS gene as evidenced from RT-PCR analyses (semi-quantitative). The maximum transcript accumulation of this gene was observed in flower petals, indicating that the petals of J. sambac flower play an important role as a major contributor of volatile precursors. The transcripts accumulation of JsDXR and JsTPS in different developmental stages and in different floral part showed that emissions of terpenoid volatiles in J. sambac flower are partially regulated at transcription levels.

  15. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma

    PubMed Central

    Gómez-Gómez, Lourdes; Parra-Vega, Verónica; Rivas-Sendra, Alba; Seguí-Simarro, Jose M.; Molina, Rosa Victoria; Pallotti, Claudia; Rubio-Moraga, Ángela; Diretto, Gianfranco; Prieto, Alicia; Ahrazem, Oussama

    2017-01-01

    Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined “crocinoplast”) were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), carotenoid isomerase (CRTISO), Crocetin glucosyltransferase 2 (UGT2), etc.) and crocin (e.g., ζ-carotene desaturase (ZDS) and plastid-lipid-associated proteins (PLAP2)) accumulation; in addition, candidate aldehyde dehydrogenase (ADH) genes were highlighted. PMID:28045431

  16. Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

    PubMed

    Yan, Ning; Zhang, Hongbo; Zhang, Zhongfeng; Shi, John; Timko, Michael P; Du, Yongmei; Liu, Xinmin; Liu, Yanhua

    2016-11-15

    Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

  17. 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.

  18. 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.

  19. [Reconstruction of muscle glycogen phosphorylase b from an apoenzyme and pyridoxal-5'-phosphate and its analogs. Interaction of apophosphorylase and the reconstructed enzyme with specific ligands].

    PubMed

    Chebotareva, N A; Sugrobova, N P; Bulanova, L N; Poznanskaia, A A; Kurganov, B I; Gunar, V I

    1995-12-01

    Sedimentation methods were used to study the effects of modification of the pyridoxal-5'-phosphate (PLP) molecule at the 5th position on the affinity of reconstituted muscle glycogen phosphorylase b for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the enzyme capacity to association induced by AMP. Reconstituted phosphorylase b was obtained with PLP analogs containing at the 5th position -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II) or -C identical to COOH (analog III) residues. Reconstitution of phosphorylase b is accompanied by the recovery of the enzyme quaternary structure. Phosphorylase b reconstituted with PLP or analogs I, II and III is not distinguished practically from the native enzyme in its affinity for glycogen. Substitution of the native coenzyme in the phosphorylase molecule with any tested PLP analog leads to lower enzyme affinity for FMN. Microscopic dissociation constants of the FMN-enzyme complexes increase in the following order: enzyme.I < enzyme.II < enzyme.III. Phosphorylase b reconstituted with analogs I, II and III differs substantially from the native enzyme in its capacity to association in the presence of 1 mM AMP: the reconstituted enzyme is represented practically by only the tetrameric form.

  20. A novel fully validated LC-MS/MS method for quantification of pyridoxal-5'-phosphate concentrations in samples of human whole blood.

    PubMed

    Ghassabian, Sussan; Griffiths, Lyn; Smith, Maree T

    2015-09-01

    Quantification of pyridoxal-5'-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20μL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was >92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80°C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

  1. Analysis of the class I aldolase binding site architecture based on the crystal structure of 2-deoxyribose-5-phosphate aldolase at 0.99A resolution.

    PubMed

    Heine, Andreas; Luz, John G; Wong, Chi-Huey; Wilson, Ian A

    2004-10-29

    The crystal structure of the bacterial (Escherichia coli) class I 2-deoxyribose-5-phosphate aldolase (DERA) has been determined by Se-Met multiple anomalous dispersion (MAD) methods at 0.99A resolution. This structure represents the highest-resolution X-ray structure of an aldolase determined to date and enables a true atomic view of the enzyme. The crystal structure shows the ubiquitous TIM alpha/beta barrel fold. The enzyme contains two lysine residues in the active site. Lys167 forms the Schiff base intermediate, whereas Lys201, which is in close vicinity to the reactive lysine residue, is responsible for the perturbed pK(a) of Lys167 and, hence, also a key residue in the reaction mechanism. DERA is the only known aldolase that is able to use aldehydes as both aldol donor and acceptor molecules in the aldol reaction and is, therefore, of particular interest as a biocatalyst in synthetic organic chemistry. The uncomplexed DERA structure enables a detailed comparison with the substrate complexes and highlights a conformational change in the phosphate-binding site. Knowledge of the enzyme active-site environment has been the basis for exploration of catalysis of non-natural substrates and of mutagenesis of the phosphate-binding site to expand substrate specificity. Detailed comparison with other class I aldolase enzymes and DERA enzymes from different organisms reveals a similar geometric arrangement of key residues and implies a potential role for water as a general base in the catalytic mechanism.

  2. Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival.

    PubMed

    García-Alcázar, Manuel; Giménez, Estela; Pineda, Benito; Capel, Carmen; García-Sogo, Begoña; Sánchez, Sibilla; Yuste-Lisbona, Fernando J; Angosto, Trinidad; Capel, Juan; Moreno, Vicente; Lozano, Rafael

    2017-03-28

    Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the rate-limiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants.

  3. Succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-Phosphate (SAICAR) Activates Pyruvate Kinase Isoform M2 (PKM2) in Its Dimeric Form.

    PubMed

    Yan, Ming; Chakravarthy, Srinivas; Tokuda, Joshua M; Pollack, Lois; Bowman, Gregory D; Lee, Young-Sam

    2016-08-23

    Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy about how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2(G415R), a variant derived from a cancer patient, which we show by size-exclusion chromatography and small-angle X-ray scattering to be a dimer that cannot form a tetramer in solution. Although PKM2(G415R) binds to fructose 1,6-bisphosphate (FBP), unlike the wild type this PKM2 variant shows no activation by FBP. In contrast, PKM2(G415R) is activated by succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-phosphate (SAICAR), an endogenous metabolite that we previously showed correlates with an increased level of cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression.

  4. Purification, properties and in situ localization of the amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase from spinach chloroplasts.

    PubMed

    Teige, M; Melzer, M; Süss, K H

    1998-03-01

    The amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase have been purified from stroma extracts of spinach chloroplasts using ammonium sulfate fractionation and FPLC. For the native enzymes, a molecular mass of 180 kDa for epimerase and 160 kDa for transketolase was found and the molecular masses of the subunits was determined to be 23 kDa for epimerase and 74 kDa for transketolase. Protein sequencing of the purified chloroplast enzymes revealed the NH2-terminal amino acid sequences of mature epimerase (NH2-TSRVDKFSKSDIIVSP) and transketolase (NH2-AAVEALESTDTDQLVEG). The enzymic properties of both enzymes such as Km values or pH optima, were found to be very similar to those for epimerases and transketolases from other sources, including yeast and animal cells. In contrast to the light-activated enzymes of the Calvin cycle, the activity of these amphibolic enzymes was not redox-dependent. Immunogold electron microscopy on spinach leaf thin sections revealed that about 90% of the total epimerase and transketolase, and 96% of the total chloroplast H+-ATP synthase portion CF1 are associated with thylakoid membranes in situ. Ribulose-1,5-bisphosphate carboxylase/oxygenase, in contrast, was evenly distributed throughout chloroplasts. These and other results indicate that minor chloroplast enzymes are arranged in a thin layer on thylakoid membrane surfaces in vivo.

  5. 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.

  6. 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.

  7. 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

  8. Colorimetric determination of the purity of 3'-phospho adenosine 5'-phosphosulfate and natural abundance of 3'-phospho adenosine 5'-phosphate at picomole quantities.

    PubMed

    Lin, E S; Yang, Y S

    1998-11-01

    This work presents novel colorimetric methods not only to measure 3'-phospho adenosine 5'-phosphate (PAP) and 3'-phospho adenosine 5'-phosphosulfate (PAPS) in the range of picomoles, but also to determine the purity of PAPS or PAP contaminants in PAPS in the range of nanomoles. These methods exploit the availability of overexpressed phenol sulfotransferase (PST) and the fact that sulfuryl group transfer requires the use of PAP or PAPS as a cofactor or cosubstrate. Experimental results indicate that absorption at 400 nm due to the production of 4-nitrophenol (pNP) is catalyzed by PST when the sulfuryl group transfers from 4-nitrophenylsulfate (pNPS) to PAP or to 2-napthol. In the absence of an acceptor substrate, PAPS is hydrolyzed to PAP by PST and is determined by sulfation with pNPS before and after this reaction. The change of absorption of pNP at 400 nm corresponds to the amount of PAP that is hydrolyzed from PAPS. Moreover, a standard curve is constructed using authentic PAP and PAP-free PST. Furthermore, this curve is used to determine the amount of PAP in extracts of pig liver, rat liver, and Escherichia coli.

  9. Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival

    PubMed Central

    García-Alcázar, Manuel; Giménez, Estela; Pineda, Benito; Capel, Carmen; García-Sogo, Begoña; Sánchez, Sibilla; Yuste-Lisbona, Fernando J.; Angosto, Trinidad; Capel, Juan; Moreno, Vicente; Lozano, Rafael

    2017-01-01

    Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the rate-limiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants. PMID:28350010

  10. Formation of Schiff bases of O-phosphorylethanolamine and O-phospho-D,L-serine with pyridoxal 5'-phosphate. experimental and theoretical studies.

    PubMed

    Vilanova, Bartolomé; Gallardo, Jessica M; Caldés, Catalina; Adrover, Miquel; Ortega-Castro, Joaquín; Muñoz, Francisco; Donoso, Josefa

    2012-03-01

    Pyridoxal 5'-phosphate (PLP) is a B(6) vitamer acting as an enzyme cofactor in various reactions of aminoacid metabolism and inhibiting glycation of biomolecules. Nonenzymatic glycation of aminophospholipids alters the stability of lipid bilayers and cell function as a result. Similarly to protein glycation, aminophospholipid glycation initially involves the formation of a Schiff base. In this work, we studied the formation of Schiff bases between PLP and two compounds mimicking the polar head of natural aminophospholipids, namely: O-phosphorylethanolamine and O-phospho-D,L-serine. Based on the results, the pH-dependence of the microscopic constants of the two PLP-aminophosphate systems studied is identical with that for PLP-aminoacid systems. However, the rate and equilibrium formation constants for the Schiff bases of the aminophosphates are low relative to those for the aminoacids. A theoretical study by density functional theory of the formation mechanism for the Schiff bases of PLP with the two aminophospholipid analogues confirmed that the activation energy of formation of the Schiff bases is greater with aminophosphates; on the other hand, that of hydrolysis is essentially similar with aminoacids and aminophosphates.

  11. The crystal structure of the Pseudomonas dacunhae aspartate-beta-decarboxylase dodecamer reveals an unknown oligomeric assembly for a pyridoxal-5'-phosphate-dependent enzyme.

    PubMed

    Lima, Santiago; Sundararaju, Bakthavatsalam; Huang, Christina; Khristoforov, Roman; Momany, Cory; Phillips, Robert S

    2009-04-24

    The Pseudomonas dacunhael-aspartate-beta-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the beta-decarboxylation of l-aspartate to produce l-alanine and CO(2). This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an l-Asp/l-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 A resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arginine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.

  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. 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.

  14. 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

  15. 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

    2012-09-06

    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.

  16. 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.

  17. A protein tyrosine phosphatase-like inositol polyphosphatase from Selenomonas ruminantium subsp. lactilytica has specificity for the 5-phosphate of myo-inositol hexakisphosphate.

    PubMed

    Puhl, Aaron A; Greiner, Ralf; Selinger, L Brent

    2008-01-01

    Although it is becoming well known that myo-inositol polyphosphates and the enzymes involved in their metabolism play a critical role in eukaryotic systems, little is understood of their significance in prokaryotic systems. A novel protein tyrosine phosphatase (PTP)-like inositol polyphosphatase (IPPase) gene has been cloned from Selenomonas ruminantium subsp. lactilytica (phyAsrl). The deduced amino acid sequence of PhyAsrl is most similar to a PTP-like IPPase from the anaerobic bacterium S. ruminantium (35% identity), but also shows similarity (19-30% identity) to various other putative prokaryotic PTPs. Recombinant PhyAsrl could dephosphorylate myo-inositol hexakisphosphate (Ins P(6)) in vitro, and maximal activity was displayed at an ionic strength of 200 mM, a pH of 4.5, and a temperature of 55 degrees C. In order to elucidate its substrate specificity and pathway of Ins P(6) dephosphorylation, a combination of kinetic and high-performance ion-pair chromatography studies were conducted. The data indicated that PhyAsrl has a general specificity for polyphosphorylated myo-inositol substrates, but can also dephosphorylate molecules containing high energy pyrophosphate bonds in vitro. PhyAsrl is unique from other microbial IPPases in that it preferentially cleaves the 5-phosphate position of Ins P(6). Furthermore, it can produce Ins(2)P via a highly unique and ordered pathway of sequential dephosphorylation: Ins P(6), Ins(1,2,3,4,6)P(5), D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2). Finally, reverse transcription PCR was used to determine that phyAsrl is constitutively expressed, and together with bioinformatic analysis, was used to gain an understanding of its physiological significance.

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

    PubMed

    Banerjee, Aparajita; Preiser, Alyssa L; Sharkey, Thomas D

    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.

  19. 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.

  20. Expression of carotenoid biosynthetic pathway genes and changes in carotenoids during ripening in tomato (Lycopersicon esculentum).

    PubMed

    Namitha, Kanakapura Krishnamurthy; Archana, Surya Narayana; Negi, Pradeep Singh

    2011-04-01

    To study the expression pattern of carotenoid biosynthetic pathway genes, changes in their expression at different stages of maturity in tomato fruit (cv. Arka Ahuti) were investigated. The genes regulating carotenoid production were quantified by a dot blot method using a DIG (dioxigenin) labelling and detection kit. The results revealed that there was an increase in the levels of upstream genes of the carotenoid biosynthetic pathway such as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (Lyt B), phytoene synthase (PSY), phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) by 2-4 fold at the breaker stage as compared to leaf. The lycopene and β-carotene content was analyzed by HPLC at different stages of maturity. The lycopene (15.33 ± 0.24 mg per 100 g) and β-carotene (10.37 ± 0.46 mg per 100 g) content were found to be highest at 5 days post-breaker and 10 days post-breaker stage, respectively. The lycopene accumulation pattern also coincided with the color values at different stages of maturity. These studies may provide insight into devising gene-based strategies for enhancing carotenoid accumulation in tomato fruits.

  1. The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis.

    PubMed

    Hsieh, Ming-Hsiun; Goodman, Howard M

    2005-06-01

    Plant isoprenoids are synthesized via two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastid nonmevalonate pathway. The Escherichia coli IspH (LytB) protein is involved in the last step of the nonmevalonate pathway. We have isolated an Arabidopsis (Arabidopsis thaliana) ispH null mutant that has an albino phenotype and have generated Arabidopsis transgenic lines showing various albino patterns caused by IspH transgene-induced gene silencing. The initiation of albino phenotypes rendered by IspH gene silencing can arise independently from multiple sites of the same plant. After a spontaneous initiation, the albino phenotype is systemically spread toward younger tissues along the source-to-sink flow relative to the initiation site. The development of chloroplasts is severely impaired in the IspH-deficient albino tissues. Instead of thylakoids, mutant chloroplasts are filled with vesicles. Immunoblot analysis reveals that Arabidopsis IspH is a chloroplast stromal protein. Expression of Arabidopsis IspH complements the lethal phenotype of an E. coli ispH mutant. In 2-week-old Arabidopsis seedlings, the expression of 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), IspD, IspE, IspF, and IspG genes is induced by light, whereas the expression of the IspH gene is constitutive. The addition of 3% sucrose in the media slightly increased levels of DXS, DXR, IspD, IspE, and IspF mRNA in the dark. In a 16-h-light/8-h-dark photoperiod, the accumulation of the IspH transcript oscillates with the highest levels detected in the early light period (2-6 h) and the late dark period (4-6 h). The expression patterns of DXS and IspG are similar to that of IspH, indicating that these genes are coordinately regulated in Arabidopsis when grown in a 16-h-light/8-h-dark photoperiod.

  2. 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

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

    2012-11-07

    The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 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 presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation 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 circular dichroism 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.

  3. L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis.

    PubMed Central

    Liu, J Q; Dairi, T; Kataoka, M; Shimizu, S; Yamada, H

    1997-01-01

    We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction. PMID:9171400

  4. Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase from Streptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis

    PubMed Central

    Kuzuyama, Tomohisa; Takagi, Motoki; Takahashi, Shunji; Seto, Haruo

    2000-01-01

    In addition to the ubiquitous mevalonate pathway, Streptomyces sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-d-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene, dxs, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among dxs homologs from six genera. The dxs gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in Escherichia coli was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a Vmax of 370 U per mg of protein and Kms of 65 μM for pyruvate and 120 μM for d-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a Km value of 35 mM for d-glyceraldehyde. To compare the enzymatic properties of CL190 and E. coli DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties. PMID:10648511

  5. Defining critical residues for substrate binding to 1-deoxy-d-xylulose 5-phosphate synthase: Active site substitutions stabilize the pre-decarboxylation intermediate C2α-lactylthiamin diphosphate

    PubMed Central

    Kakalis, Lazaros; Jordan, Frank; Meyers, Caren L. Freel

    2014-01-01

    1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-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 uses mutagenesis and a combination of protein fluorescence, circular dichroism and kinetics experiments to investigate the roles of Arg-420, Arg-478 and Tyr-392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism and predict Arg-420 and Arg-478 are involved in binding of the acceptor substrate, d-GAP. d-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg-420 and Arg-478, also accelerates decarboxylation of the pre-decarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating this binding interaction is not absolutely required, and the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr-392 contributes to d-GAP affinity and is not required for LThDP formation or its d-GAP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate LThDP is significantly stabilized on R420A and Y392F variants compared to wild type DXP synthase in the absence of acceptor substrate, yet these substitutions do not appear to impact the rate of d-GAP-promoted LThDP decarboxylation in the presence of high d-GAP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues to promote d-GAP binding which in turn facilitates decarboxylation, and further highlight interesting differences between DXP synthase and other ThDP-dependent enzymes. PMID:24767541

  6. Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

    PubMed Central

    Blanc, V; Lagneaux, D; Didier, P; Gil, P; Lacroix, P; Crouzet, J

    1995-01-01

    In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin. PMID:7665509

  7. VOC emissions of Grey poplar leaves as affected by salt stress and different N sources.

    PubMed

    Teuber, M; Zimmer, I; Kreuzwieser, J; Ache, P; Polle, A; Rennenberg, H; Schnitzler, J-P

    2008-01-01

    Nitrogen nutrition and salt stress experiments were performed in a greenhouse with hydroponic-cultured, salt-sensitive Grey poplar (Populus x canescens) plants to study the combined influence of different N sources (either 1 mm NO(3) (-) or NH(4)(+)) and salt (up to 75 mm NaCl) on leaf gas exchange, isoprene biosynthesis and VOC emissions. Net assimilation and transpiration proved to be highly sensitive to salt stress and were reduced by approximately 90% at leaf sodium concentrations higher than 1,800 microg Na g dry weight (dw)(-1). In contrast, emissions of isoprene and oxygenated VOC (i.e. acetaldehyde, formaldehyde and acetone) were unaffected. There was no significant effect of combinations of salt stress and N source, and neither NO(3)(-) or NH(4)(+) influenced the salt stress response in the Grey poplar leaves. Also, transcript levels of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PcDXR) and isoprene synthase (PcISPS) did not respond to the different N sources and only responded slightly to salt application, although isoprene synthase (PcISPS) activity was negatively affected at least in one of two experiments, despite high isoprene emission rates. A significant salt effect was the strong reduction of leaf dimethylallyl diphosphate (DMADP) content, probably due to restricted availability of photosynthates for DMADP biosynthesis. Further consequences of reduced photosynthetic gas exchange and maintaining VOC emissions are a very high C loss, up to 50%, from VOC emissions related to net CO(2) uptake and a strong increase in leaf internal isoprene concentrations, with maximum mean values up to 6.6 microl x l(-1). Why poplar leaves maintain VOC biosynthesis and emission under salt stress conditions, despite impaired photosynthetic CO(2) fixation, is discussed.

  8. The role of phosphate in a multistep enzymatic reaction: reactions of the substrate and intermediate in pieces.

    PubMed

    Kholodar, Svetlana A; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

    2015-02-25

    Several mechanistically unrelated enzymes utilize the binding energy of their substrate's nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-D-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-C-methyl-D-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme-phosphodianion interactions to the reaction coordinate.

  9. Comparative glandular trichome transcriptome based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

    PubMed

    Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

    2017-02-11

    The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome was carried out. In addition to the MVA and MEP pathway genes, about 210 and 196 different terpene synthases (TPS) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (3 from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. Abbreviations - AACT, acetyl-CoA C-acetyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; DGE, digital gene expression; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FPPS, farnesyl pyrophosphate synthase; GC, gas chromatography; GPPS, geranyl pyrophosphate synthase; GT, glandular trichome; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; HMGS, hydroxymethylglutaryl-CoA synthase; IDI, isopentenyl-diphosphate delta-isomerase; IPD, isopiperitenol dehydrogenase; IPI

  10. Chemical synthesis of long RNAs with terminal 5'-phosphate groups.

    PubMed

    Pradère, Ugo; Halloy, François; Hall, Jonathan

    2017-03-12

    Long structured RNAs are useful biochemical and biological tools. They are usually prepared enzymatically, but this precludes their site-specific modification with functional groups for chemical biology studies. One solution is to perform solid-phase synthesis of multiple RNAs loaded with 5'-terminal phosphate groups, so that RNAs can be concatenated using template ligation reactions. However, there are currently no readily available reagents suitable for the incorporation of the phosphate group into long RNAs by solid-phase synthesis. Here we describe an easy-to-prepare phosphoramidite reagent suitable for the chemical introduction of 5'-terminal phosphate groups into long RNAs. The phosphate is protected by a dinitrobenzhydryl group that serves as an essential lipophilic group for the separation of oligonucleotide by-products. The phosphate is unmasked quantitatively by brief UV irradiation. We demonstrate the value of this reagent in the preparation of a library of long structured RNAs that are site-specifically modified with functional groups.

  11. Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5'-phosphates by mammalian phosphotransferases.

    PubMed

    Feng, Joy Y; Parker, William B; Krajewski, Megan L; Deville-Bonne, Dominique; Veron, Michel; Krishnan, Preethi; Cheng, Yung-Chi; Borroto-Esoda, Katyna

    2004-11-01

    Amdoxovir [(-)-beta-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5'-nucleotidase (5'-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM). Human 5'-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP. In CEM cells incubated with 5 microM DXG for 24 h, 0.015 pmole/10(6) cells (approximately 7.5 nM) of DXG-TP was detected as the primary metabolite. Our study demonstrated that 5'-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.

  12. 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

  13. A Critical Examination of the Reaction of Pyridoxal 5-Phosphate with Human Hemoglobin Ao

    DTIC Science & Technology

    1989-01-01

    Second, extracellular hemoglobin is quickly lost from the vascular system (vascular half-life - 3 h ), which is generally considered due to the...20,000 x g for 1 h . To avoid contamination with stroma, only the upper two-thirds of this solution was removed and passed through a mixed-bed ion...autoburette. Water’- saturated air or nitrogen was then flushed over the surface for periods of up to 4 h at room temperature. Deoxygenation was

  14. Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from Selenomonas ruminantium

    PubMed Central

    Sagong, Hye-Young; Son, Hyeoncheol Francis; Kim, Sunghwan; Kim, Yong-Hwan; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-01-01

    Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family. PMID:27861532

  15. The Role of the Phosphatidylinositol-5-Phosphate 4-Kinases in p53-Null Breast Cancers

    DTIC Science & Technology

    2015-10-01

    mutant   backgrounds...aberrations  in  p53.   Targeting  these  enzymes  with  novel  agents  might  prevent  the  growth  of  p53   mutant ...PIP4K2A  and/or   PIP4K2B  would  be  an  effective  therapy  for  TP53   mutant  breast  cancers.  A  second

  16. Interaction of muscle glycogen phosphorylase b reconstituted from apoenzyme and analogs of pyridoxal-5'-phosphate with specific ligands.

    PubMed

    Chebotareva, N A; Sugrobova, N P; Bulanova, L N; Poznanskaya, A A; Kurganov, B I; Gunar, V I

    1996-04-01

    Phosphorylase b from rabbit skeletal muscles was reconstituted with analogs of PLP containing residues -CH(2)-CH(2)-COOH, trans-CH=CH-COOH or -C=-COOH at position 5. Replacing native coenzyme in the phosphorylase molecule with any PLP analog tested leads to the decrease in the enzyme affinity for the allosteric inhibitor, FMN. Phosphorylase b reconstituted with analogs of PLP shows the greater ability for association in tetramers in the presence of 1 mM AMP than native enzyme.

  17. Structure-activity relationships of compounds targeting mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.

    PubMed

    Mao, Jialin; Eoh, Hyungjin; He, Rong; Wang, Yuehong; Wan, Baojie; Franzblau, Scott G; Crick, Dean C; Kozikowski, Alan P

    2008-10-01

    We report on a target-based approach to identify possible Mycobacterium tuberculosis DXS inhibitors from the structure of a known transketolase inhibitor. A small focused library of analogs was assembled in order to begin elucidating some meaningful structure-activity relationships of 3-(4-chloro-phenyl)-5-benzyl-4H-pyrazolo[1,5-a]pyrimidin-7-one. Ultimately we found that 2-methyl-3-(4-fluorophenyl)-5-(4-methoxy-phenyl)-4H-pyrazolo[1,5-a]pyrimidin-7-one, although still weak, was able to inhibit M. tuberculosis DXS with an IC(50) of 10.6 microM.

  18. 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

  19. Biosynthesis of benzofuran derivatives in root cultures of Tagetes patula via phenylalanine and 1-deoxy-D-xylulose 5-phosphate.

    PubMed

    Margl, Lilla; Ettenhuber, Christian; Gyurján, István; Zenk, Meinhart H; Bacher, Adelbert; Eisenreich, Wolfgang

    2005-04-01

    Root cultures of Tagetes patula L. cv. Carmen were grown with a mixture of unlabeled glucose and [U-(13)C(6)]glucose or [1-(13)C(1)]glucose as carbon source. Isoeuparin and (-)-4-hydroxytremetone were isolated by solvent extraction of the cultured tissue, purified by chromatography and analysed by (1)H and (13)C NMR spectroscopy. Amino acids obtained by hydrolysis of protein from the same experiments were used for the reconstruction of the labelling patterns in central metabolic intermediates. These labelling patterns were used for the prediction of isotopolog compositions in the benzofuranone derivatives via different hypothetical pathways. Comparison with the experimentally observed isotopolog distributions showed that the benzenoid ring and the acetoxy group are exclusively or predominantly (>98%) derived from phenylalanine and not from acetyl-CoA via a polyketide-type biosynthesis. The isopropylidene side chain and two carbon atoms of the furan and dihydrofuran moiety, respectively, originate from an isoprenoid building block obtained exclusively or predominantly (>98%) via the deoxyxylulose phosphate pathway. The exomethylene atom of the isopropylidene side chain is biosynthetically equivalent to the (Z)-methyl group of dimethylallyl diphosphate. The data indicate that isoeuparin and (-)-4-hydroxytremetone are assembled from 4-hydroxyacetophenone and dimethylallyl diphosphate via prenyl-substituted 4-hydroxyacetophenone and dihydrobenzofurans as intermediates.

  20. A second target of the antimalarial and antibacterial agent fosmidomycin revealed by cellular metabolic profiling†

    PubMed Central

    Zhang, Baichen; Watts, Kristin M.; Hodge, Dana; Kemp, Lisa M.; Hunstad, David A.; Hicks, Leslie M.; Odom, Audrey R.

    2011-01-01

    Antimicrobial drug resistance is an urgent problem in control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum, and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies to treat malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and E. coli bacteria: in both organisms, metabolic profiling demonstrated a block in isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin, but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent to block non-mevalonate isoprenoid metabolism, and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse

  1. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    SciTech Connect

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; Davis, Ryan W.

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.

  2. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    DOE PAGES

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; ...

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

  3. Circadian Rhythms of Isoprene Biosynthesis in Grey Poplar Leaves1

    PubMed Central

    Loivamäki, Maaria; Louis, Sandrine; Cinege, Gyöngyi; Zimmer, Ina; Fischbach, Robert J.; Schnitzler, Jörg-Peter

    2007-01-01

    Isoprene (2-methyl-1,3-butadiene) emission varies diurnally in different species. In poplar (Populus spp.), it has recently been shown that the gene encoding the synthesizing enzyme for isoprene, isoprene synthase (ISPS), displays diurnal variation in expression. Working on shoot cultures of Grey poplar (Populus × canescens) placed under a different light regime in phytochambers, we showed that these variations in PcISPS gene expression, measured by quantitative real-time polymerase chain reaction, are not only due to day-night changes, but also are linked to an internal circadian clock. Measurement of additional selected isoprenoid genes revealed that phytoene synthase (carotenoid pathway) displays similar fluctuations, whereas 1-deoxy-d-xylulose 5-phosphate reductoisomerase, possibly the first committed enzyme of the 1-deoxy-d-xylulose 5-phosphate pathway, only shows light regulation. On the protein level, it appeared that PcISPS activity and protein content became reduced under constant darkness, whereas under constant light, activity and protein content of this enzyme were kept high. In contrast, isoprene emission rates under continuous irradiation displayed circadian changes as is the case for gene expression of PcISPS. Furthermore, binding assays with Arabidopsis (Arabidopsis thaliana) late elongated hypocotyl, a transcription factor of Arabidopsis involved in circadian regulation, clearly revealed the presence of circadian-determining regulatory elements in the promoter region of PcISPS. PMID:17122071

  4. Cloning and functional analysis of the orotidine-5'-phosphate decarboxylase gene (PbrURA3) of the pathogenic fungus Paracoccidioides brasiliensis.

    PubMed

    Reinoso, Cristina; Sorais, Françoise; Niño-Vega, Gustavo A; Fermiñán, Encarnación; San-Blas, Gioconda; Domínguez, Angel

    2005-07-15

    A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Erdosteine prevents doxorubicin-induced cardiotoxicity in rats.

    PubMed

    Yagmurca, Murat; Fadillioglu, Ersin; Erdogan, Hasan; Ucar, Muharrem; Sogut, Sadik; Irmak, M Kemal

    2003-10-01

    The clinical use of doxorubicin (Dxr) is limited by its cardiotoxic effects which are mediated by oxygen radicals. The purpose of this study was to investigate in vivo protective effects of erdosteine, an antioxidant agent because of its secondary active metabolites in vivo, against the cardiotoxicity induced by Dxr in rats. Three groups of male Sprague-Dawley rats (60 days old) were used. Group 1 was untreated group used as control; the other groups were treated with Dxr (single i.p. dosage of 20 mg kg(-1) b.wt.) or Dxr plus erdosteine (10 mg kg(-1) day(-1), orally), respectively. Erdosteine or oral saline treatment was done starting 2 days before Dxr for 12 days. The analyses were done at the 10th day of Dxr treatment. The protein carbonyl content, the activities of myeloperoxidase, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) as well as heart rate and blood pressures were significantly increased in Dxr group in comparison with the other groups. However, pulse pressure was decreased in Dxr group. The body and heart weights were decreased in both Dxr administered groups in comparison with control group. Disorganization of myocardial histology, picnotic nuclei, edema, and increase in collagen content around vessels were seen in the slides of Dxr group, whereas normal myocardial microscopy was preserved in Dxr plus erdosteine group. Collectively, these in vivo hemodynamic, enzymatic and morphologic studies provide an evidence for a possible prevention of cardiac toxicity in Dxr-treated patients.

  14. Protective effects of erdosteine against doxorubicin-induced cardiomyopathy in rats.

    PubMed

    Fadillioğlu, Ersin; Erdoğan, Hasan; Söğüt, Sadik; Kuku, Irfan

    2003-01-01

    The usefulness of doxorubicin (DXR) is limited by its cardiotoxicity. In order to improve future DXR therapy by using a new antioxidant agent, an experimental study was designed. This study was undertaken to determine whether DXR-induced cardiotoxicity is prevented by erdosteine, a mucolytic agent showing antioxidant properties. Three groups of male Sprague-Dawley rats (60 days old) were used: one group was untreated as a control; the other groups were treated with DXR (single i.p. dosage of 20 mg kg(-1) body wt.) or DXR plus erdosteine (10 mg kg(-1) day(-1), orally), respectively. The DXR treatment without erdosteine increased antioxidant enzyme activities and also increased lipid peroxidation in myocardial tissue. The rats treated with DXR plus erdosteine produced a significant decrease in lipid peroxidation in comparison with control and DXR groups. Furthermore, erdosteine administration led to an increase in antioxidant enzyme activities in comparison with the control group. Erdosteine treatment also increased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in comparison with the DXR group. There was no significant difference in lipid peroxidation of myocardial tissue between control and DXR plus erdosteine-treated rats. It was concluded that erdosteine caused an increase in the activities of antioxidant enzymes, especially GSH-Px and CAT, protecting the heart tissue sufficiently from oxidative damage to membrane lipids and other cellular components induced by DXR.

  15. 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

  16. Regulator of G Protein Signaling 6 (RGS6) Mediates Doxorubicin-induced ATM and p53 Activation by a Reactive Oxygen Species (ROS)-dependent Mechanism

    PubMed Central

    Huang, Jie; Yang, Jianqi; Maity, Biswanath; Mayuzumi, Daisuke; Fisher, Rory A.

    2011-01-01

    Doxorubicin (DXR), among the most widely used cancer chemotherapy agents, promotes cancer cell death via activation of ATM and the resultant up-regulation of tumor suppressor p53. The exact mechanism by which DXR activates ATM is not fully understood. Here we discovered a novel role for Regulator of G protein Signaling 6 (RGS6) in mediating activation of ATM and p53 by DXR. RGS6 was robustly induced by DXR, and genetic loss of RGS6 dramatically impaired DXR-induced activation of ATM and p53, as well as its in vivo apoptotic actions in heart. The ability of RGS6 to promote p53 activation in response to DXR was independent of RGS6 interaction with G proteins but required ATM. RGS6 mediated activation of ATM and p53 by DXR via a ROS-dependent and DNA damage-independent mechanism. This mechanism represents the primary means by which DXR promotes activation of the ATM-p53-apoptosis pathway that underlies its cytotoxic activity. Our findings contradict the canonical theories that DXR activates ATM primarily by promoting DNA damage either directly or indirectly (via ROS) and that RGS6 function is mediated by its interactions with G proteins. These findings reveal a new mechanism for the chemotherapeutic actions of DXR and identify RGS6 as a novel target for cancer chemotherapy. PMID:21859827

  17. Effects of erdosteine treatment against doxorubicin-induced toxicity through erythrocyte and plasma oxidant/antioxidant status in rats.

    PubMed

    Fadillioğlu, Ersin; Erdoğan, Hasan

    2003-04-01

    The clinical use of doxorubicin (Dxr), an antineoplastic agent, is limited by its extensive toxicity which is mostly mediated by oxidant injury. We have studied the effect of erdosteine, a mucolytic drug showing antioxidant properties, in preventing Dxr-toxicity to improve future Dxr therapy. Male Sprague-Dawley rats were divided into four groups. The first group that underwent no medication was accepted as control group; the second group was treated with a single i.p. injection of Dxr (20 mg kg(-1) b.wt.); the third group was treated with oral erdosteine alone (10 mg kg(-1) b.wt. day(-1) for 12 days); and in the last group erdosteine was administered starting before Dxr injection for 12 days. The thiobarbituric acid reactive substances (TBARS) level of Dxr group was higher in both plasma and erythrocyte than the other groups. In plasma and erythrocyte, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased in Dxr plus erdosteine group in comparison with control group, and the activities of GSH-Px were increased in Dxr plus erdosteine group in comparison with Dxr group. The erythrocyte catalase (CAT) activity of Dxr plus erdosteine group was higher than control and Dxr groups. Plasma xanthine oxidase activities and nitric oxide (NO) levels were not significantly different between groups, however erythrocyte NO level of Dxr group was higher than control. In conclusion, Dxr administration resulted in increased lipid peroxidation in plasma as well as erythrocyte and erdosteine treatment helped to prevent oxidative injury by increasing antioxidant enzymes, especially SOD, GSH-Px and CAT, in rats.

  18. Dalbergioidin Ameliorates Doxorubicin-Induced Renal Fibrosis by Suppressing the TGF-β Signal Pathway

    PubMed Central

    Ren, Xianguo; Bo, Yun; Fan, Junting; Chen, Maosheng; Xu, Daliang; Dong, Yang; He, Haowei; Ren, Xianzhi; Qu, Rong; Jin, Yulian

    2016-01-01

    We investigated the effect of Dalbergioidin (DAL), a well-known natural product extracted from Uraria crinita, on doxorubicin- (DXR-) induced renal fibrosis in mice. The mice were pretreated for 7 days with DAL followed by a single injection of DXR (10 mg/kg) via the tail vein. Renal function was analyzed 5 weeks after DXR treatment. DXR caused nephrotoxicity. The symptoms of nephrotic syndrome were greatly improved after DAL treatment. The indices of renal fibrosis, the phosphorylation of Smad3, and the expression of alpha-smooth muscle actin (α-SMA), fibronectin, collagen III (Col III), E-cadherin, TGF-β, and Smad7 in response to DXR were all similarly modified by DAL. The present findings suggest that DAL improved the markers for kidney damage investigated in this model of DXR-induced experimental nephrotoxicity. PMID:28100935

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Amelioration of Doxorubicin-Induced Cardiac and Renal Toxicity by Oxycarotenoid Lutein and Its Mechanism of Action.

    PubMed

    Sindhu, Edakkadath Raghavan; Nithya, Thattaruparambil Raveendran; Binitha, Ponnamparambil Purushothaman; Kuttan, Ramadasan

    2016-01-01

    We set out to determine the effect of oxycarotenoid lutein on reducing cardiac and renal toxicity induced by doxorubicin (DXR). We started with oral administration in rats of lutein for 15 d before administering DXR (30 mg/kg body weight, intraperitoneally, in a single dose). Animals in all groups were sacrificed 24 h after DXR administration. Serum markers of cardiac injury lactate dehydrogenase, creatine phosphokinase, serum glutamate oxaloacetate transaminase, and serum glutamate pyruvate transaminase increased drastically after DXR but decreased after lutein treatment (p < 0.001). Elevated serum urea and creatinine in DXR-treated rats were reduced by lutein treatment (p < 0.001). Lutein increased superoxide dismutase, catalase, glutathione peroxidase, and glutathione levels in cardiac and renal tissues of DXR-treated rats. Pretreatment of lutein reduced DXR-induced rise of oxidative stress markers including lipid peroxidation, tissue hydroperoxides, and conjugated dienes in cardiac and renal tissue. These findings were supported by electrocardiogram measurements and histopathological analyses. Results confirmed the protection of lutein against cardiac and renal toxicity induced by DXR in rats.

  4. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

    PubMed

    Jadaun, Jyoti Singh; Sangwan, Neelam S; Narnoliya, Lokesh K; Singh, Neha; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh

    2017-04-01

    Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system.

  5. 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.

  6. The Carotenogenesis Pathway via the Isoprenoid-β-carotene Interference Approach in a New Strain of Dunaliella salina Isolated from Baja California Mexico

    PubMed Central

    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 β-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 β-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-β-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 µM fosmidomycin, carotenogenesis and the synthesis of β-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 β-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. PMID:19370170

  7. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants.

    PubMed

    Cordoba, Elizabeth; Salmi, Mari; León, Patricia

    2009-01-01

    The methyl-D-erythritol 4-phosphate pathway is responsible for the biosynthesis of a substantial number of natural compounds of biological and biotechnological importance. In recent years, this pathway has become an obvious target to develop new herbicides and antimicrobial drugs. In addition, the production of a variety of compounds of medical and agricultural interest may be possible through the genetic manipulation of this pathway. To this end, a complete understanding of the molecular mechanisms that regulate this pathway is of tremendous importance. Recent data have accumulated that show some of the multiple mechanisms that regulate the methyl-D-erythritol 4-phosphate pathway in plants. In this review we will describe some of these and discuss their implications. It has been demonstrated that 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of this route, plays a major role in the overall regulation of the pathway. A small gene family codes for this enzyme in most of the plants which have been analysed so far, and the members of these gene families belong to different phylogenetic groups. Each of these genes exhibits a distinct expression pattern, suggesting unique functions. One of the most interesting regulatory mechanisms recently described for this pathway is the post-transcriptional regulation of the level of DXS and DXR proteins. In the case of DXS, this regulation appears conserved among plants, supporting its importance. The evidence accumulated suggests that this regulation might link the activity of this pathway with the plant's physiological conditions and the metabolic demand for the final products of this route.

  8. 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.

  9. 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.

  10. Doxorubicin-induced alopecia is associated with sebaceous gland degeneration.

    PubMed

    Selleri, Silvia; Seltmann, Holger; Gariboldi, Silvia; Shirai, Yuri F; Balsari, Andrea; Zouboulis, Christos C; Rumio, Cristiano

    2006-04-01

    Alopecia, accompanied by skin dryness, is one of the distressing side effects often occurring in chemotherapy-treated cancer patients. Little is known of the effects of chemotherapy on sebaceous glands, despite their importance in hair follicle homeostasis. This study investigates sebaceous gland morphology and the response of SZ95 sebaceous gland cell line to doxorubicin (DXR) treatment. The morphology of sebaceous glands during intraperitoneal DXR treatment was investigated by optical and electron microscopy in a 7-day-old rat model and further confirmed in an adult mouse model. Moreover, in vitro studies using the SZ95 sebaceous gland cell line were performed to assess the response of sebocytes to DXR in terms of cell proliferation, apoptosis, and necrosis. DXR treatment induced sebaceous gland regression and occasionally caused their complete disappearance. This observed damage and disappearance preceded DXR-induced hair loss. In vitro experiments using the SZ95 sebaceous gland cell line indicated that DXR treatment induced a differentiation process leading to premature sebocytes apoptosis. Owing to the importance of the sebaceous gland in hair follicle homeostasis, DXR-induced involution of this gland might be related to subsequent hair loss.

  11. Usefulness of hexamethylenetetramine in combination with chemotherapy using free and pegylated liposomal doxorubicin in vivo, referring to the effect on quiescent cells.

    PubMed

    Masunaga, Shin-Ichiro; Kono, Kenji; Nakamura, Jun; Tano, Keizo; Yoshida, Hiroyuki; Watanabe, Masami; Kashino, Genro; Suzuki, Minoru; Kinashi, Yuko; Liu, Yong; Ono, Koji

    2009-05-01

    SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They received hexamethylenetetramine (HMTA) either once intraperitoneally or continuously subcutaneously together with chemotherapy using intraperitoneally administered free doxorubicin (DXR) or intravenously injected pegylated liposomal doxorubicin (PLD). One hour after the free DXR loading or 24 h after the PLD loading, the response of intratumor quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cell population was determined from the tumors not treated with BrdU. Encapsulation of DXR into pegylated liposomes significantly enhanced cytotoxicity, especially in Q cells. HMTA, especially when administered continuously, efficiently increased the sensitivity to DXR, particularly in Q cells. The increase in sensitivity on the continuous rather than single administration of HMTA was a little clearer in the total cell population than in Q cells. DXR's encapsulation into pegylated liposomes and combination with HMTA, particularly when administered continuously, apparently reduced the difference in sensitivity to free DXR between the total and Q cell populations. In terms of the tumor cell-killing effect as a whole, including Q cells, the encapsulation of DXR into pegylated liposomes and combination with HMTA, particularly through continuous administration, are very promising, taking into account that HMTA has been used clinically.

  12. Protective effects of the exopolysaccharide Lasiodiplodan against DNA damage and inflammation induced by doxorubicin in rats: Cytogenetic and gene expression assays.

    PubMed

    Mello, M B; Machado, C S; Ribeiro, D L; Aissa, A F; Burim, R V; Alves da Cunha, M A; Barcelos, G R M; Antunes, L M G; Bianchi, M L P

    2017-02-01

    The lasiodiplodan (LS) is a β-(1→6)-d-glucan produced by the fungus Lasiodiplodia theobromae and some of the biological activities of LS were reported as hypoglycemic, anticoagulant, anti-proliferative and anticancer action; however, its effects on DNA instability and modulation of gene expression are still unclear. Aims of study were investigate the genotoxic effects of lasiodiplodan, and its protective activity against DNA damage induced by doxorubicin (DXR) and its impact on the expression of genes associated with DNA damage and inflammatory response pathways. Therefore, Wistar rats were treated (15 days) orally with LS (5.0; 10 and 20mg/kg bw) alone and in combination with DXR (15mg/kg bw; administrated intraperitoneally on 14th day) as well as their respective controls: distilled water and DXR. Monitoring of DNA damage was assessed by comet and micronucleus (MN) assays and gene expression was evaluated by PCR-Arrays. Treatments with LS alone did not induce disturbances on DNA; when LS was given in combination with DXR, comet and MN formations were reduced to those found in the respective controls. Moreover, LS was able to reduce the disturbances on gene expressions induced by DXR treatment, since the animals that receive LS associated with DXR showed no alteration in the expression of genes related to DNA damage response. Also, DXR induced several up- and down-regulation of several genes associated to inflammatory process, while the animals that received LS+DXR had their gene expression patterns similar to those found in the control group. In conclusion, our results showed that LS did not induce disturbances on DNA stability and significantly reduce the DNA damage and inflammation caused by DXR exposure. In addition, we give further information concerning the molecular mechanisms associated to LS protective effects which seems to be a promising nutraceutical with chemopreventive potential.

  13. Transcription of the protein kinase C-δ gene is activated by JNK through c-Jun and ATF2 in response to the anticancer agent doxorubicin

    PubMed Central

    Min, Byong Wook; Kim, Chang Gun; Ko, Jesang; Lim, Yoongho

    2008-01-01

    Expression of protein kinase C-δ (PKCδ) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKCδ gene transcription. In the present study, we demonstrate that JNK stimulates PKCδ gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKCδ promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKCδ promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKCδ gene expression. PMID:19116455

  14. Antimutagenic and antirecombinagenic activities of noni fruit juice in somatic cells of Drosophila melanogaster.

    PubMed

    Franchi, Leonardo P; Guimarães, Nilza N; De Andrade, Laise R; De Andrade, Heloísa H R; Lehmann, Maurício; Dihl, Rafael R; Cunha, Kênya S

    2013-01-01

    Noni, a Hawaiian name for the fruit of Morinda citrifolia L., is a traditional medicinal plant from Polynesia widely used for the treatment of many diseases including arthritis, diabetes, asthma, hypertension and cancer. Here, a commercial noni juice (TNJ) was evaluated for its protective activities against the lesions induced by mitomycin C (MMC) and doxorrubicin (DXR) using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for two genetic markers (mwh and flr3 ), were co-treated with TNJ plus MMC or DXR. We have observed a reduction in genotoxic effects of MMC and DXR caused by the juice. TNJ provoked a marked decrease in all kinds of MMC- and DXR-induced mutant spots, mainly due to its antirecombinagenic activity. The TNJ protective effects were concentration-dependent, indicating a dose-response correlation, that can be attributed to a powerful antioxidant and/or free radical scavenger ability of TNJ.

  15. Beta-cyclodextrin conjugates with glucose moieties designed as drug carriers: their syntheses, evaluations using concanavalin A and doxorubicin, and structural analyses by NMR spectroscopy.

    PubMed

    Oda, Yoshiki; Kobayashi, Natsumi; Yamanoi, Takashi; Katsuraya, Kaname; Takahashi, Keiko; Hattori, Kenjiro

    2008-05-01

    Three kinds of beta-cyclodextrin derivatives conjugated with glucose moieties, which were expected as models for a drug carrier targeting the drug delivery systems, were designed and synthesized from beta-cyclodextrin and the natural product, 4-hydroxyphenyl-beta-D-glucopyranoside called arbutin. Arbutin was used because it had a phenyl group with a hydroxyl function which could be used to link the glucose moiety to beta-cyclodextrin. The evaluations of these conjugates as the drug-carrying molecules were done by investigating the molecular interactions with the carbohydrate-binding Concanavalin A (Con A) lectin and the anticancer agent, doxorubicin (DXR), using an SPR optical biosensor. The association constants of the conjugates with immobilized Con A were 2.0 x 10(3) approximately 8.8 x 10(3) M(-1). The result showed that the Con A bound to the glucose moieties from arbutin in the conjugates with prospective association constants. The inclusion associations of the conjugates with immobilized DXR reached 2.2 x 10(5) approximately 1.4 x 10(8) M(-1). The extremely high inclusion associations for DXR suggested their potential abilities as drug-carrying molecules for carrying DXR. The NMR analyses indicated that the phenyl group of the conjugates greatly served to increase the inclusion associations for DXR. In their DXR inclusion complexes, the formation of the stacking complexes by the pi;-pi interactions between the phenyl groups and the included DXR also enhanced their inclusion abilities for DXR.

  16. pH-Responsive Theranostic Polymer-Caged Nanobins (PCNs): Enhanced Cytotoxicity and T1 MRI Contrast by Her2-Targeting

    PubMed Central

    Hong, Bong Jin; Swindell, Elden P.; MacRenaris, Keith W.; Hankins, Patrick L.; Chipre, Anthony J.; Mastarone, Daniel J.; Ahn, Richard W.; Meade, Thomas J.; O’Halloran, Thomas V.

    2014-01-01

    A PCN theranostic platform comprises a doxorubicin (DXR)-loaded liposomal core and an acid-sensitive polymer shell that is functionalized with Herceptin and GdIII-based MRI contrast agents. In vitro testing reveals a 14-fold increase in DXR-based cytotoxicity versus a non-targeted analogue and an 120-fold improvement in cellular GdIII–uptake in comparison with clinically approved DOTA-GdIII, leading to significant T1 MRI contrast enhancement. PMID:24516291

  17. 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.

  18. Chemotherapy-Induced Late Transgenerational Effects in Mice

    PubMed Central

    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.

    2011-01-01

    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. PMID:21437292

  19. Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance

    PubMed Central

    Avnet, Sofia; Lemma, Silvia; Cortini, Margherita; Pellegrini, Paola; Perut, Francesca; Zini, Nicoletta; Kusuzaki, Katsuyuki; Chano, Tokuhiro; Grisendi, Giulia; Dominici, Massimo; De Milito, Angelo; Baldini, Nicola

    2016-01-01

    Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24–48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, short-term acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance. PMID:27566564

  20. 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.

  1. 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.

  2. Potential sources of quantification error when retrospectively assessing metacarpal bone loss from historical radiographs by using digital X-ray radiogrammetry: an experimental study.

    PubMed

    Kälvesten, Johan; Brismar, Torkel B; Persson, Anders

    2014-01-01

    During the past 15 yr, digital X-ray radiogrammetry (DXR) has been used to measure metacarpal bone mineral density (BMD). BMD is often measured in existing cohorts where X-ray images were not acquired in accordance with the DXR imaging protocol (DIP). The purpose of the present study was to analyze how deviations from DIP in historical radiographs may affect the reproducibility of DXR-BMD measurements. Cadaver hand phantoms were used to conduct repeat measurements of deviations from DIP with respect to voltage, exposure, lateral displacement, supination, combination of lateral displacement and supination or rotation, extension of the wrist, and edge enhancement. Direct digital radiography (Aristos; Siemens Healthcare, Erlangen, Germany) was used for image acquisition and dxr-online (Sectra, Linköping, Sweden) for DXR-BMD measurements. The influence of the tested deviations from DIP ranged from 0 to 32.5 mg/cm(2) (0-6.8%). On repetition with the same specimen, none of the deviations resulted in a within-specimen reproducibility error greater than 2 mg/cm(2) (0.4%, equivalent to a T-score of 0.042). Among the tested deviations, all except tube voltage had a magnitude greater than the normal measurement noise for the technique and must therefore be considered when planning a study based on historical images.

  3. The Streptomyces peucetius dpsY and dnrX Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis

    PubMed Central

    Lomovskaya, Natalia; Doi-Katayama, Yukiko; Filippini, Sylvia; Nastro, Cecilia; Fonstein, Leonid; Gallo, Mark; Colombo, Anna Luisa; Hutchinson, C. Richard

    1998-01-01

    The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria. PMID:9573189

  4. 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

  5. Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

    PubMed Central

    Peiris, Diluka; Spector, Alexander F.; Lomax-Browne, Hannah; Azimi, Tayebeh; Ramesh, Bala; Loizidou, Marilena; Welch, Hazel; Dwek, Miriam V.

    2017-01-01

    Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer. PMID:28223691

  6. Fasting regulates EGR1 and protects from glucose- and dexamethasone-dependent sensitization to chemotherapy

    PubMed Central

    Vinciguerra, Manlio; Rappa, Francesca; Wei, Min; Brandhorst, Sebastian; Cappello, Francesco; Mirzaei, Hamed; Lee, Changhan; Longo, Valter D.

    2017-01-01

    Fasting reduces glucose levels and protects mice against chemotoxicity, yet drugs that promote hyperglycemia are widely used in cancer treatment. Here, we show that dexamethasone (Dexa) and rapamycin (Rapa), commonly administered to cancer patients, elevate glucose and sensitize cardiomyocytes and mice to the cancer drug doxorubicin (DXR). Such toxicity can be reversed by reducing circulating glucose levels by fasting or insulin. Furthermore, glucose injections alone reversed the fasting-dependent protection against DXR in mice, indicating that elevated glucose mediates, at least in part, the sensitizing effects of rapamycin and dexamethasone. In yeast, glucose activates protein kinase A (PKA) to accelerate aging by inhibiting transcription factors Msn2/4. Here, we show that fasting or glucose restriction (GR) regulate PKA and AMP-activated protein kinase (AMPK) to protect against DXR in part by activating the mammalian Msn2/4 ortholog early growth response protein 1 (EGR1). Increased expression of the EGR1-regulated cardioprotective peptides atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in heart tissue may also contribute to DXR resistance. Our findings suggest the existence of a glucose–PKA pathway that inactivates conserved zinc finger stress-resistance transcription factors to sensitize cells to toxins conserved from yeast to mammals. Our findings also describe a toxic role for drugs widely used in cancer treatment that promote hyperglycemia and identify dietary interventions that reverse these effects. PMID:28358805

  7. 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

  8. Protective effects of proanthocyanidins of grape (Vitis vinifera L.) seeds on DNA damage induced by Doxorubicin in somatic cells of Drosophila melanogaster.

    PubMed

    de Rezende, Alexandre Azenha Alves; Graf, Ulrich; Guterres, Zaira da Rosa; Kerr, Warwick Estevam; Spanó, Mário Antônio

    2009-07-01

    Proanthocyanidins (PAs), also known as condensed tannins, are naturally occurring oligomers and polymers of flavan-3-ol monomer units widely found in the leaves, flowers, fruits, seeds, nuts and barks of many plants. Grape seed proanthocyanidins (GSPs) have been used as nutritional supplements, as antioxidants, in preventing atherosclerosis and cardiovascular diseases, and for dislipidemy treatment. The anthracycline antibiotic adriamycin (Doxorubicin, DXR) is a cancer chemotherapeutic agent that interferes with the topoisomerase II enzyme and generates free radicals. In the present study, GSPs (1.680, 3.375, or 6.750 mg/mL) alone were examined for genotoxicity, and combined with DXR (0.125 mg/mL) for antigenotoxicity, using the standard (ST) and high bioactivation (HB) versions of the wing somatic mutation and recombination test in Drosophila melanogaster. The results observed in both crosses were rather similar. GSPs themselves did not show genotoxicity at the doses used. GSPs suppressed the DNA damage induced by DXR in a dose-dependent manner. Comparison of the frequencies of wing spots in the marker-heterozygous (MH) flies and balancer-heterozygous (BH) flies from both crosses, indicated that induced recombination was the major response for the treatments with DXR alone. The co-treatments demonstrated that GSPs have some anti-mutagenic activity; however, anti-recombinagenic activity was the major response.

  9. Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

    PubMed Central

    Baek, NamHuk; Seo, Ok Won; Kim, MinSung; Hulme, John; An, Seong Soo A

    2016-01-01

    Recently, increasing numbers of cell culture experiments with 3D spheroids presented better correlating results in vivo than traditional 2D cell culture systems. 3D spheroids could offer a simple and highly reproducible model that would exhibit many characteristics of natural tissue, such as the production of extracellular matrix. In this paper numerous cell lines were screened and selected depending on their ability to form and maintain a spherical shape. The effects of increasing concentrations of doxorubicin (DXR) on the integrity and viability of the selected spheroids were then measured at regular intervals and in real-time. In total 12 cell lines, adenocarcinomic alveolar basal epithelial (A549), muscle (C2C12), prostate (DU145), testis (F9), pituitary epithelial-like (GH3), cervical cancer (HeLa), HeLa contaminant (HEp2), embryo (NIH3T3), embryo (PA317), neuroblastoma (SH-SY5Y), osteosarcoma U2OS, and embryonic kidney cells (293T), were screened. Out of the 12, 8 cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U2OS formed regular spheroids and the effects of DXR on these structures were measured at regular intervals. Finally, 5 cell lines, A549, HeLa, SH-SY5Y, U2OS, and 293T, were selected for real-time monitoring and the effects of DXR treatment on their behavior were continuously recorded for 5 days. A potential correlation regarding the effects of DXR on spheroid viability and ATP production was measured on days 1, 3, and 5. Cytotoxicity of DXR seemed to occur after endocytosis, since the cellular activities and ATP productions were still viable after 1 day of the treatment in all spheroids, except SH-SY5Y. Both cellular activity and ATP production were halted 3 and 5 days from the start of the treatment in all spheroids. All cell lines maintained their spheroid shape, except SHSY-5, which behaved in an unpredictable manner when exposed to toxic concentrations of DXR. Cytotoxic effects of DXR towards SH-SY5Y seemed to cause degradation of

  10. [Kinetics and equilibrium of reactions between nucleotides and methylol derivatives of beta-alanine].

    PubMed

    Khulordava, K G; Kosaganov, Iu N; Lazurkin, Iu S

    1978-01-01

    The rate constants of forward and reverse reactions between methylol derivatives of beta-alanine and deoxycytidine 5'-phosphate, deoxyadenosine 5'phosphate and deoxyguanosine 5'phosphate and the equilibrium constants of these reactions were determined by the spectrophotometric method at 39,5 degrees C and pH 6,95. Besides, the equilibrium constant of the reaction between beta-alanine and formaldehyde was determined. Unlike deoxycytidine and deoxyadenosine 5'-phosphates, interaction of deoxyguanosine 5'phosphate with methylol derivatives is more complicated. A model proposed for the interaction of deoxyguanosine 5'phosphate with methylol derivatives explains the behavior of this nucleotide in the reaction. The kinetic and equilibrium constants of the interaction of methylol derivatives with nucleotides investigated exceed by two or three orders of magnitude the corresponding constants of the interaction of formaldehyde with these nucleotides.

  11. Pyridine analogs inhibit the glucosyltransferase of Streptococcus mutans.

    PubMed Central

    Thaniyavarn, S; Taylor, K G; Singh, S; Doyle, R J

    1982-01-01

    Soluble glucan synthesis catalyzed by dextransucrase preparations from Streptococcus mutans 6715 were inhibited by pyridoxal-5-phosphate and several other pyridine analogs, including pyridoxine, pyridoxamine, pyridoxamine-5-phosphate, pyridoxal, and 4-pyridoxic acid. Pyridine and pyridine-4-carboxaldehyde were not effective inhibitors of the enzyme. Kinetic analyses suggested that pyridoxal-5-phosphate is a noncompetitive inhibitor of dextransucrase. The inactivation was dependent on time, pyridoxal-5-phosphate concentration, and hydrogen ion concentration. Apparent Ki values were 4.9 mM at pH 7.0 and 4.2 mM at pH 5.5. Dextransucrase activity could be restored by dialysis to remove the inhibitors. Maximum inhibition was observed after a 120-min incubation of the enzyme with pyridoxal-5-phosphate. The pH optima for inhibition by pyridoxal-5-phosphate were 4 and 7. The sucrose-dependent adherence of S. mutans cells to saliva-coated hydroxylapatite beads was also inhibited by pyridoxal-5-phosphate but only marginally by the other pyridine anatogs. In addition, pyridoxal-5-phosphate markedly reduced the rate of acid production by intact S. mutans cells from sucrose or glucose substrates. Another pyridoxal-5-phosphate analog, 2-methyl-5-hydroxypyridine, was also effective in preventing the production of acid by S. mutans from sucrose or glucose. When S. mutans cells were preincubated with pyridoxal-5-phosphate or pyridine analogs, significant reductions in the rate of D-glucose uptake were observed. It is suggested that the inhibition of dextransucrase occurs because of a change iun enzyme conformation which results from the binding of the pyridine derivatives. The results suggest that pyridoxal-5-phosphate or structural analogs may ultimately be useful in reducing the incidence of dental caries. PMID:6215355

  12. 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.

  13. Comparison between Radionuclide Ventriculography and Echocardiography for Quantification of Left Ventricular Systolic Function in Rats Exposed to Doxorubicin

    PubMed Central

    de Oliveira, Luciano Fonseca Lemos; O'Connell, João Lucas; de Carvalho, Eduardo Elias Vieira; Pulici, Érica Carolina Campos; Romano, Minna Moreira Dias; Maciel, Benedito Carlos; Simões, Marcus Vinicius

    2017-01-01

    Background Radionuclide ventriculography (RV) is a validated method to evaluate the left ventricular systolic function (LVSF) in small rodents. However, no prior study has compared the results of RV with those obtained by other imaging methods in this context. Objectives To compare the results of LVSF obtained by RV and echocardiography (ECHO) in an experimental model of cardiotoxicity due to doxorubicin (DXR) in rats. Methods Adult male Wistar rats serving as controls (n = 7) or receiving DXR (n = 22) in accumulated doses of 8, 12, and 16 mg/kg were evaluated with ECHO performed with a Sonos 5500 Philips equipment (12-MHz transducer) and RV obtained with an Orbiter-Siemens gamma camera using a pinhole collimator with a 4-mm aperture. Histopathological quantification of myocardial fibrosis was performed after euthanasia. Results The control animals showed comparable results in the LVSF analysis obtained with ECHO and RV (83.5 ± 5% and 82.8 ± 2.8%, respectively, p > 0.05). The animals that received DXR presented lower LVSF values when compared with controls (p < 0.05); however, the LVSF values obtained by RV (60.6 ± 12.5%) were lower than those obtained by ECHO (71.8 ± 10.1%, p = 0.0004) in this group. An analysis of the correlation between the LVSF and myocardial fibrosis showed a moderate correlation when the LVSF was assessed by ECHO (r = -0.69, p = 0.0002) and a stronger correlation when it was assessed by RV (r = -0.79, p < 0.0001). On multiple regression analysis, only RV correlated independently with myocardial fibrosis. Conclusion RV is an alternative method to assess the left ventricular function in small rodents in vivo. When compared with ECHO, RV showed a better correlation with the degree of myocardial injury in a model of DXR-induced cardiotoxicity. PMID:28146205

  14. Changes in P-glycoprotein activity are mediated by the growth of a tumour cell line as multicellular spheroids

    PubMed Central

    Valeria, Ponce de León; Raúl, Barrera-Rodríguez

    2005-01-01

    Background Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to multidrug resistance in tumours. However, the physiological role of P-gp in tumours growing as multicellular spheroids is not well understood. Recent evidence suggests that P-gp activity may be modulated by cellular components such as membrane proteins, membrane-anchoring proteins or membrane-lipid composition. Since, multicellular spheroids studies have evidenced alterations in numerous cellular components, including those related to the plasma membrane function, result plausible that some of these changes might modulate P-gp function and be responsible for the acquisition of multicellular drug resistance. In the present study, we asked if a human lung cancer cell line (INER-51) grown as multicellular spheroids can modify the P-gp activity to decrease the levels of doxorubicin (DXR) retained and increase their drug resistance. Results Our results showed that INER-51 spheroids retain 3-folds lower doxorubicin than the same cells as monolayers however; differences in retention were not observed when the P-gp substrate Rho-123 was used. Interestingly, neither the use of the P-gp-modulating agent cyclosporin-A (Cs-A) nor a decrease in ATP-pools were able to increase DXR retention in the multicellular spheroids. Only the lack of P-gp expression throughout the pharmacological selection of a P-gp negative (P-gpneg) mutant clone (PSC-1) derived from INER-51 cells, allow increase of DXR retention in spheroids. Conclusion Thus, multicellular arrangement appears to alter the P-gp activity to maintain lower levels of DXR. However, the non expression of P-gp by cells forming multicellular spheroids has only a minor impact in the resistance to chemotherapeutic agents. PMID:16001980

  15. Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein.

    PubMed

    Villar, Victor Hugo; Vögler, Oliver; Barceló, Francisca; Gómez-Florit, Manuel; Martínez-Serra, Jordi; Obrador-Hevia, Antònia; Martín-Broto, Javier; Ruiz-Gutiérrez, Valentina; Alemany, Regina

    2014-04-01

    The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10-100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3 ± 1.11% and 68.8 ± 1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5-10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.

  16. Research of Extension of the Life Cycle of Helicopter Rotor Blade in Hungary

    DTIC Science & Technology

    2003-02-01

    Radiography (DXR), and (iii) Vibration Diagnostics (VD) with Statistical Energy Analysis (SEA) were semi- simultaneously applied [1]. The used three...2.2. Vibration Diagnostics (VD)) Parallel to the NDT measurements the Statistical Energy Analysis (SEA) as a vibration diagnostical tool were...noises were analysed with a dual-channel real time frequency analyser (BK2035). In addition to the Statistical Energy Analysis measurement a small

  17. Increase in E-selectin expression in umbilical vein endothelial cells by anticancer drugs and inhibition by cimetidine.

    PubMed

    Kawase, Jin; Ozawa, Soji; Kobayashi, Kenichi; Imaeda, Yoshihiro; Umemoto, Shunji; Matsumoto, Sumio; Ueda, Masakazu

    2009-12-01

    E-selectin is expressed on the surfaces of stimulated vascular endothelial cells and is sometimes involved in cancer cell metastasis. The H2-receptor antagonist cimetidine inhibits the increase in E-selectin expression on vascular endothelial cells that is induced by interleukin-1beta (IL-1beta) and cimetidine. It also inhibits the adhesion of sialyl-Lewis-antigen-positive cancer cells to vascular endothelial cells, ultimately inhibiting hematogenous metastasis. Anticancer drugs are essential to cancer therapy, but whether they can alter the expression of E-selectin in vascular endothelial cells remains unclear. Whether cimetidine inhibits the expression of E-selectin in the same manner in the presence or absence of anticancer drugs also remains unknown. Human umbilical vein endothelial cells were cultured with 5-fluorouracil (5-FU), doxorubicin (DXR), cisplatin (CDDP), or IL-1beta and with or without cimetidine. The expression of E-selectin at the mRNA and protein levels was then determined using quantitative reverse transcription-polymerase chain reaction and immunohistochemical staining, respectively. The E-selectin mRNA level increased in cells exposed to 5-FU, DXR, or CDDP, but the addition of cimetidine had no effect on the E-selectin mRNA level. The expression of E-selectin protein was also significantly higher after the addition of 5-FU, DXR, or CDDP, compared with that of a negative control. However, when cimetidine was added prior to the addition of 5-FU, DXR, or CDDP, the expression of E-selectin was significantly suppressed. Thus, cimetidine significantly inhibited the expression of E-selectin at the protein level without affecting its expression at the mRNA level in cells treated with anticancer drugs. In conclusion, anticancer drugs increased the expression of E-selectin and this increase was inhibited by cimetidine. These findings suggest that the administration of cimetidine during treatment with anticancer drugs might be useful for preventing

  18. 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.

  19. Underwater Nondestructive Testing of Ship Hull Welds

    DTIC Science & Technology

    1979-09-01

    5. cowt * "n’d Mar(itini A~niamiitrati~on W.. P. N. UZD, Wr. P., a. XWLRA Dxr~etor Chiorf Brwmoh of M2rine 011 Butt iht~itwgL Dip"Jon and Gas ...Radiography, ultrasonic inspection, and magnetic particle testing are discussed including the modifications necessary for underwater applications . In... Measuring Weld Reinforcement 7 3 Recommended Positioning of Electrical Prods When Inspecting Butt Welds 7 4 An Example of Crack Detection Using

  20. Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius.

    PubMed

    Malla, Sailesh; Niraula, Narayan Prasad; Liou, Kwangkyoung; Sohng, Jae Kyung

    2009-08-01

    To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

  1. Therapeutic efficacy of the combination of doxorubicin-loaded liposomes with inertial cavitation generated by confocal ultrasound in AT2 Dunning rat tumour model.

    PubMed

    Mestas, Jean-Louis; Fowler, R Andrew; Evjen, Tove J; Somaglino, Lucie; Moussatov, Alexei; Ngo, Jacqueline; Chesnais, Sabrina; Røgnvaldsson, Sibylla; Fossheim, Sigrid L; Nilssen, Esben A; Lafon, Cyril

    2014-09-01

    The combination of liposomal doxorubicin (DXR) and confocal ultrasound (US) was investigated for the enhancement of drug delivery in a rat tumour model. The liposomes, based on the unsaturated phospholipid dierucoylphosphocholine, were designed to be stable during blood circulation in order to maximize accumulation in tumour tissue and to release drug content upon US stimulation. A confocal US setup was developed for delivering inertial cavitation to tumours in a well-controlled and reproducible manner. In vitro studies confirm drug release from liposomes as a function of inertial cavitation dose, while in vivo pharmacokinetic studies show long blood circulation times and peak tumour accumulation at 24-48 h post intravenous administration. Animals injected 6 mg kg(-1) liposomal DXR exposed to US treatment 48 h after administration show significant tumour growth delay compared to control groups. A liposomal DXR dose of 3 mg kg(-1), however, did not induce any significant therapeutic response. This study demonstrates that inertial cavitation can be generated in such a fashion as to disrupt drug carrying liposomes which have accumulated in the tumour, and thereby increase therapeutic effect with a minimum direct effect on the tissue. Such an approach is an important step towards a therapeutic application of cavitation-induced drug delivery and reduced chemotherapy toxicity.

  2. Anti-invasive adjuvant therapy with imipramine blue enhances chemotherapeutic efficacy against glioma.

    PubMed

    Munson, Jennifer M; Fried, Levi; Rowson, Sydney A; Bonner, Michael Y; Karumbaiah, Lohitash; Diaz, Begoña; Courtneidge, Sara A; Knaus, Ulla G; Brat, Daniel J; Arbiser, Jack L; Bellamkonda, Ravi V

    2012-03-28

    The invasive nature of glioblastoma (GBM) represents a major clinical challenge contributing to poor outcomes. Invasion of GBM into healthy tissue restricts chemotherapeutic access and complicates surgical resection. Here, we test the hypothesis that an effective anti-invasive agent can "contain" GBM and increase the efficacy of chemotherapy. We report a new anti-invasive small molecule, Imipramine Blue (IB), which inhibits invasion of glioma in vitro when tested against several models. IB inhibits NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-mediated reactive oxygen species generation and alters expression of actin regulatory elements. In vivo, liposomal IB (nano-IB) halts invasion of glioma, leading to a more compact tumor in an aggressively invasive RT2 syngeneic astrocytoma rodent model. When nano-IB therapy was followed by liposomal doxorubicin (nano-DXR) chemotherapy, the combination therapy prolonged survival compared to nano-IB or nano-DXR alone. Our data demonstrate that nano-IB-mediated containment of diffuse glioma enhanced the efficacy of nano-DXR chemotherapy, demonstrating the promise of an anti-invasive compound as an adjuvant treatment for glioma.

  3. 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.

  4. Antigenotoxicity of artepillin C in vivo evaluated by the micronucleus and comet assays.

    PubMed

    de Azevedo Bentes Monteiro Neto, Moacir; de Souza Lima, Ildercílio Mota; Furtado, Ricardo Andrade; Bastos, Jairo Kenupp; da Silva Filho, Ademar Alves; Tavares, Denise Crispim

    2011-11-01

    Artepillin C (3,5-diprenyl-p-coumaric acid), a major compound found in Brazilian green propolis and Baccharis dracunculifolia, shows anti-inflammatory, antibacterial, antiviral, antioxidant and antitumoral activities, among others. The aim of this study was to evaluate the genotoxic potential of artepillin C and its ability to prevent the chemically induced chromosome breakage or loss and the primary DNA damage using the micronucleus and comet assays in male Swiss mice, respectively. The animals were treated by gavage with different doses of artepillin C (0.4, 0.8 and 1.6 mg kg(-1) b.w.). For the antigenotoxicity assays, the different doses of artepillin C were administered simultaneously to doxorubicin (DXR; micronucleus test; 15 mg kg(-1) b.w.) and to methyl methanesulfonate (MMS; comet assay; 40 mg kg(-1) b.w.). The results showed that artepillin C itself was not genotoxic in the mouse micronucleus and comet assays. In the animals treated with artepillin C and DXR, the number of micronucleated reticulocytes was significantly lower in comparison with the animals treated only with DXR. Regarding antigenotoxicity, artepillin C at the tested doses significantly reduced the extent of DNA damage in liver cells induced by MMS.

  5. Chemical modification of lysine and arginine residues of bovine heart 2-oxoglutarate dehydrogenase: effect on the enzyme activity and regulation.

    PubMed

    Ostrovtsova, S A

    1998-01-01

    Chemical modification of arginine and lysine residues of bovine heart 2-oxoglutarate dehydrogenase with phenylglyoxal and pyridoxal 5'-phosphate inactivated the enzyme, indicating the importance of these residues for the catalysis. Inactivation caused by pyridoxal 5'-phosphate was prevented in the presence of thiamine pyrophosphate and Mg2+ allowing the assumption that lysine residues participate in binding of the cofactor.

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

    DOEpatents

    Buelter, Thomas [Denver, CO; Meinhold, Peter [Denver, CO; Feldman, Reid M. Renny [San Francisco, CA; Hawkins, Andrew C [Parker, CO; Urano, Jun [Irvine, CA; Bastian, Sabine [Pasadena, CA; Arnold, Frances [La Canada, CA

    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.

  7. Methods for library-scale computational protein design.

    PubMed

    Johnson, Lucas B; Huber, Thaddaus R; Snow, Christopher D

    2014-01-01

    Faced with a protein engineering challenge, a contemporary researcher can choose from myriad design strategies. Library-scale computational protein design (LCPD) is a hybrid method suitable for the engineering of improved protein variants with diverse sequences. This chapter discusses the background and merits of several practical LCPD techniques. First, LCPD methods suitable for delocalized protein design are presented in the context of example design calculations for cellobiohydrolase II. Second, localized design methods are discussed in the context of an example design calculation intended to shift the substrate specificity of a ketol-acid reductoisomerase Rossmann domain from NADPH to NADH.

  8. 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.

  9. In vivo Studies on the Protective Effect of Propolis on Doxorubicin-Induced Toxicity in Liver of Male Rats

    PubMed Central

    Singla, Shivani; Kumar, Neelima R.; Kaur, Jaspreet

    2014-01-01

    Objective: Since anticancer drugs are to be administered for long durations of time and are associated with systemic toxicities, the present studies were conducted to evaluate the protective potential of honey bee propolis against a widely used anticancer drug, doxorubicin (DXR) induced toxicity and oxidative damage in liver tissues of rats. Materials and Methods: Sixteen male Sprague Dawley rats, weighing between 200-220 g, were used and were divided into four equal groups. Propolis was given orally to rats [250 mg/kg body weight (bw) for 14 consecutive days] and DXR [25 mg/kg bw; intraperitoneally (i.p) was administered on 12th, 13th and 14th day of the experiment. All the animals were sacrificed on day 15th day by decapitation. Blood and tissue samples were collected for measurement of toxicity and oxidative damage parameters (enzymatic assays and biochemical estimations). Results: Administration of DXR for 3 days at a cumulative dose of 25 mg/kg bw, induced toxicity and oxidative stress in rats as significantly decreased activity of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were observed in rat liver supernatants when compared to control group. Increased activity of serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) was obtained in DXR administered rats. Also there are significantly increased levels of lipid peroxides (measured as malondialdehyde formation) and significantly decreased level of glutathione (GSH) in doxorubicin treated rat liver supernatants as compared to healthy controls. On the other hand, administration of animals with propolis prior to DXR treatment led to significant modulation of the oxidative damage related parameters in liver and hepatotoxicity parameters in blood, when compared to doxorubicin treated group. However results were still not comparable to control group or only propolis group indicating

  10. Purification and Characterization of Pea Chloroplastic Phosphoriboisomerase 12

    PubMed Central

    Skrukrud, Cynthia L.; Gordon, Ilana M.; Dorwin, Sally; Yuan, Xiao-Hua; Johansson, Göte; Anderson, Louise E.

    1991-01-01

    Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase. ImagesFigure 1 PMID:16668459

  11. 40 CFR 63.601 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... part 60, appendix A, Method 5. Phosphate rock calciner means the equipment used to remove moisture and.... Phosphate rock feed means all material entering any phosphate rock dryer or phosphate rock...

  12. 40 CFR 63.601 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... part 60, appendix A, Method 5. Phosphate rock calciner means the equipment used to remove moisture and.... Phosphate rock feed means all material entering any phosphate rock dryer or phosphate rock...

  13. 40 CFR 63.601 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... part 60, appendix A, Method 5. Phosphate rock calciner means the equipment used to remove moisture and.... Phosphate rock feed means all material entering any phosphate rock dryer or phosphate rock...

  14. 40 CFR 63.601 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... part 60, appendix A, Method 5. Phosphate rock calciner means the equipment used to remove moisture and.... Phosphate rock feed means all material entering any phosphate rock dryer or phosphate rock...

  15. 40 CFR 63.601 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... part 60, appendix A, Method 5. Phosphate rock calciner means the equipment used to remove moisture and.... Phosphate rock feed means all material entering any phosphate rock dryer or phosphate rock...

  16. Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation.

    PubMed

    Sottani, Cristina; Rinaldi, Paola; Leoni, Emanuela; Poggi, Guido; Teragni, Cristina; Delmonte, Angelo; Minoia, Claudio

    2008-09-01

    A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.

  17. Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

    PubMed

    Pinzón-Daza, Martha L; Cuellar-Saenz, Yenith; Nualart, Francisco; Ondo-Mendez, Alejandro; Del Riesgo, Lilia; Castillo-Rivera, Fabio; Garzón, Ruth

    2017-01-20

    P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 9999: 1-11, 2017. © 2017 Wiley Periodicals, Inc.

  18. A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni).

    PubMed

    Kumar, Hitesh; Kaul, Kiran; Bajpai-Gupta, Suphla; Kaul, Vijay Kumar; Kumar, Sanjay

    2012-01-15

    Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA(3)) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.

  19. Evaluation of hand bone loss by digital X-ray radiogrammetry as a complement to clinical and radiographic assessment in early rheumatoid arthritis: results from the SWEFOT trial

    PubMed Central

    2013-01-01

    Background To investigate hand bone loss (HBL) measured by digital X-ray radiogrammetry (DXR) in patients with early rheumatoid arthritis (RA) receiving different treatment regimens, and to evaluate if DXR change rates during the first 12 months correlate with radiological damage after 24 months. Methods From the total SWEFOT trial population, 159 patients had hand radiographs correctly timed and taken with same modality to be analyzed with DXR. All patients started treatment with methotrexate. After 3–4 months, patients with DAS28 > 3.2 were randomized to add sulfasalazine and hydroxychloroquine (triple therapy) or infliximab (MTX + INF). Those with DAS28 ≤3.2 were followed in regular care. Radiographic progression over 24 months was scored according to the Sharp van der Heijde score (SHS) and defined as >5 increase in T-SHS over 24 months. Hand bone mineral density (BMD) was measured by DXR at inclusion and 12 months and a change ≥2.5 mg/cm2/month was used as a cut-off for HBL. Results In the MTX responders, triple therapy, and MTX + INF groups, the proportions with HBL were 4.1%, 22.2% and 16.4%, respectively (p = 0.01), and the mean (SD) radiological progression in these groups was 3.91 (6.72), 7.40 (14.63) and 2.72 (4.55) respectively (p = 0.06). Patients with HBL had significantly greater risk for radiographic progression, compared with patients without HBL (odds ratio 3.09, 95% CI =1.20–7.79, p = 0.02). Conclusions Non-responders to MTX had a significantly greater risk of HBL than MTX-responders, despite the add-on therapies. Patients with HBL during the 12 months had greater risk of radiographic progression after 24 months. Evaluation of HBL may help to identify patients who are at risk of radiographic progression. PMID:23497111

  20. 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.

  1. Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves.

    PubMed Central

    Schnarrenberger, C.; Flechner, A.; Martin, W.

    1995-01-01

    The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP. PMID:12228497

  2. 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

  3. Genetic modification of alphaGal expression in xenogeneic endothelial cells yields a complex immunological response.

    PubMed

    Fischbeck, J A; Baier, J M; Akella, R; Hern-Anderson, D; Schmidt, C E

    2001-12-01

    The source of cells for tissue engineering applications remains a hurdle, predominantly for procedures in which there is insufficient time to harvest a patient's own cells. Animal cells are readily available, but undergo immune rejection. Rejection of animal (i.e., xenogeneic) tissue involves practically every component of the immune system. The initial phase, hyperacute rejection (HAR), involves natural xenoreactive antibodies and the complement system, and leads to endothelial cell lysis and rapid tissue destruction. The cell-surface epitope, galactose-alpha(1,3)-galactose (alphaGal), is presumed to play a key role in HAR. The later stage of immune response (delayed xenograft rejection or DXR), is mediated by immune cells such as monocytes. Carbohydrates are likely also involved in DXR, but their role in this phase of the immune response is less clear. A better understanding of all stages of xenogeneic immune rejection may make it feasible to create cell lines that are immune tolerant. In these studies, we have genetically modified bovine endothelial cells to study the roles of carbohydrates in immune rejection. Our studies suggest that one or more epitopes other than alphaGal may influence complement-mediated lysis. Furthermore, antibodies, as instigators in the complement response, and monocytes appear to recognize different cell surface epitopes.

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

    PubMed

    de Sousa, Neila C; de Rezende, Alexandre A A; da Silva, Regildo M G; Guterres, Zaira R; Graf, Ulrich; Kerr, Warwick E; Spanó, Mário A

    2009-04-01

    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.

  5. ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

    PubMed Central

    Andrisse, Stanley; Patel, Gaytri D.; Chen, Joseph E.; Webber, Andrea M.; Spears, Larry D.; Koehler, Rikki M.; Robinson-Hill, Rona M.; Ching, James K.; Jeong, Imju; Fisher, Jonathan S.

    2013-01-01

    Objective The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1. Research Design and Methods L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity. Results In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL. Conclusions These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction. PMID:23776597

  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. 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

  8. An assessment of GafChromic film for measuring 50 kV and 100 kV percentage depth dose curves.

    PubMed

    Fletcher, Claire Lesley; Mills, John A

    2008-06-07

    Percentage depth dose (PDD) curves were obtained for 50 kV and 100 kV x-rays on a Gulmay Medical D3000 DXR unit. Different dosimetry systems were compared including a Scanditronix Wellhofer small volume cylindrical ion chamber, a Wellhofer photon PFD diode, a PTW soft x-ray parallel plate chamber (N23342) and two types of radiochromic film: GafChromic EBT and GafChromic MD55. The PDD curves were also compared to BEAMnrc Monte Carlo predictions. GafChromic film was found to be a valid choice of dosimeter for measuring percentage depth dose curves at 100 kV and 50 kV. All the dosimeters showed agreement with predictions at depths greater than 10 mm, while near the surface GafChromic film and PFD diodes give the best agreement to Monte Carlo values.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26291039

  10. 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.

  11. Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues.

    PubMed

    Kubota, T; Furukawa, T; Tanino, H; Suto, A; Otan, Y; Watanabe, M; Ikeda, T; Kitajima, M

    2001-01-01

    Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc.htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

  12. Salinomycin induces cell death via inactivation of Stat3 and downregulation of Skp2.

    PubMed

    Koo, K H; Kim, H; Bae, Y-K; Kim, K; Park, B-K; Lee, C-H; Kim, Y-N

    2013-06-27

    Salinomycin has been shown to control breast cancer stem cells, although the mechanisms underlying its anticancer effects are not clear. Deregulation of cell cycle regulators play critical roles in tumorigenesis, and they have been considered as anticancer targets. In this study, we investigated salinomycin effect on cell cycle progression using OVCAR-8 ovarian cancer cell line and multidrug-resistant NCI/ADR-RES and DXR cell lines that are derived from OVCAR-8. Parental OVCAR-8 cells are sensitive to several anticancer drugs, but NCI/ADR-RES and DXR cells are resistant to several anticancer drugs. However, salinomycin caused cell growth inhibition and apoptosis via cell cycle arrest at G1 in all three cell lines. Salinomycin inhibited signal transducer and activator of transcription 3 (Stat3) activity and thus decreased expression of Stat3-target genes, including cyclin D1, Skp2, and survivin. Salinomycin induced degradation of Skp2 and thus accumulated p27Kip1. Knockdown of Skp2 further increased salinomycin-induced G1 arrest, but knockdown of p27Kip1 attenuated salinomycin effect on G1 arrest. Cdh1, an E3 ligase for Skp2, was shifted to nuclear fractions upon salinomycin treatment. Cdh1 knockdown by siRNA reversed salinomycin-induced Skp2 downregulation and p27Kip1 upregulation, indicating that salinomycin activates the APC(Cdh1)-Skp2-p27Kip1 pathway. Concomitantly, si-Cdh1 inhibited salinomycin-induced G1 arrest. Taken together, our data indicate that salinomycin induces cell cycle arrest and apoptosis via downregulation or inactivation of cell cycle-associated oncogenes, such as Stat3, cyclin D1, and Skp2, regardless of multidrug resistance.

  13. 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...

  14. 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; ...

  15. 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...

  16. 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...

  17. Enzymatic Basis for d-Arabitol Production by Saccharomyces rouxii1

    PubMed Central

    Ingram, Jordan M.; Wood, W. A.

    1965-01-01

    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-C14 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 (NADPH2) and d-xylulose with reduced nicotinamide adenine dinucleotide (NADH2). 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 NADH2 was purified 33-fold and characterized as a xylitol (→ d-xylulose) dehydrogenase. Similarly, an enzyme reducing d-ribulose with NADPH2 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 NADPH2-linked d-arabitol (→ d-ribulose) dehydrogenase. PMID:14292984

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

    PubMed Central

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

    2011-01-01

    Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-d-ribose 5-phosphate (ribose 5-phosphate) and α-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 α-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 Mn2+-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-[18O3]phosphate and [U-13C5]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. PMID:21193409

  19. Lifetime Prediction of Organic Coating/Metal System.

    DTIC Science & Technology

    1980-08-01

    210 5. Phosphate Conversion Coatings ....... . ..... .. .. . .... .......... 26 a. Deposition Kinetics...samples ............. 25 13 Potential-time curves for steel of four different surface pretreatments immersed in phosphating solution...28 14 Surface morphology of zinc phosphate conversion coating on steel for five different pretreatment procedures ............... 30 15 Typical

  20. Biochemical retrosynthesis of 2'-deoxyribonucleosides 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

    2006-08-01

    2'-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2'-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2'-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2'-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker's yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5'-triphosphate generated from adenosine 5'-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via D-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2'-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2'-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2'-deoxyribonucleoside synthesis. 2'-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.

  1. 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.

  2. 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.

  3. [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.

  4. Polyphosphorylation and non-enzymatic template-directed ligation of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Oligonucleotide 5'-polyphosphates are formed under potentially prebiotic conditions from oligonucleotide 5'-phosphates and sodium trimetaphosphate. Oligonucleotides activated as polyphosphates undergo template-directed ligation. We believe that these reactions could have produced longer oligonucleotide products from shorter substrates under prebiotic conditions.

  5. 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.

  6. 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

  7. Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

    PubMed Central

    Ray, P H

    1980-01-01

    3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate. PMID:6988389

  8. 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

  9. Oxidative stress proteins as an indicator of a low quality of eucalyptus clones for the pulp and paper industry.

    PubMed

    Britto, D S; Pirovani, C P; Gonzalez, E R; Silva, J F; Gesteira, A S; Cascardo, J C M

    2012-10-19

    Eucalyptus is a genus widely cultivated in many tropical and subtropical regions of the world as one of the main sources of raw materials for the pulp and paper industry. Identification of clones and selection of genotypes with desirable agronomic characteristics would be useful. We assessed eucalyptus full-sibs that varied in wood quality, using a combination of two-dimensional gel electrophoresis and mass spectrometry to identify differentially expressed proteins as candidates for quality markers. Thirty-one differently expressed proteins were identified, including three proteins of clone X1, four of clone X2, and 12 each of clones X3 and X4. These proteins are involved in various biological processes, including polyphosphate biosynthesis, catalytic activity, nucleotide excision repair, cellular metabolic processes, cell redox homeostasis, response to salt stress, response to temperature, oxidation and reduction processes, cellular water homeostasis, and protein phosphorylation. In the cambial region of each clone, the proteins ketol-acid reductoisomerase, uncharacterized protein MG428, receptor-like serine/threonine-protein kinase and a heat shock protein were found in larger quantities in clone X4 than in clone X1. These proteins are known to be related to protection against oxidative stress and biosynthesis of lignin. A high buildup of proteins involved in response to stress in the cambial region of eucalyptus would indicate clones with undesirable characteristics for use in the pulp and paper industry.

  10. 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

  11. 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.

  12. A proteomic analysis of salt stress response in seedlings of two African rice cultivars.

    PubMed

    Damaris, Rebecca Njeri; Li, Ming; Liu, Yanli; Chen, Xi; Murage, Hunja; Yang, Pingfang

    2016-11-01

    Salt stress is one of the key abiotic stresses threatening future agricultural production and natural ecosystems. This study investigates the salt stress response of two rice seedlings, which were screened from 28 Kenya rice cultivars. A proteomic analysis was carried out and Mapman bin codes employed in protein function categorization. Proteins in the redox, stress, and signaling categories were identified, and whose expression differed between the salt tolerant and the salt sensitive samples employed in the present study. 104 and 102 root proteins were observed as significantly altered during salt stress in the tolerant and sensitive samples, respectively and 13 proteins were commonly expressed. Among the 13 proteins, ketol-acid reductoisomerase protein was upregulated in both 1 and 3days of salt treatment in the tolerant sample, while it was down-regulated in both 1 and 3days of salt treatment in the sensitive sample. Actin-7, tubulin alpha, V-type proton ATPase, SOD (Cu-Zn), SOD (Mn), and pyruvate decarboxylase were among the observed salt-induced proteins. In general, this study improves our understanding about salt stress response mechanisms in rice.

  13. 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

  14. 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.

  15. DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage.

    PubMed

    Suji, G; Sivakami, S

    2007-11-01

    Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.

  16. The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli.

    PubMed Central

    Ploux, O; Marquet, A

    1992-01-01

    The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5'-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5'-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM. Images Fig. 1. PMID:1575677

  17. Regulation of Photosynthetic Capacity in Chlamydomonas mundana 1

    PubMed Central

    Russell, George K.; Gibbs, Martin

    1966-01-01

    A regulatory system has been described in the obligately phototrophic green alga Chlamydomonas mundana. Cells grown in acetate media are unable to fix carbon dioxide in the light but carry out a photoassimilation of acetate to carbohydrate: cells cultured with carbon dioxide as the sole source of cellular carbon carry out typical green plant photosynthesis. The control appears to take place at the level of the reductive pentose phosphate cycle. The presence of sodium acetate in the medium strongly inhibits formation of ribulose-1.5-diphosphate carboxylase, ribulose-5-phosphate kinase, and one of the 2 fructose-1,6-diphosphate aldolase activities of the cell. Ribose-5-phosphate isomerase is present in higher activity in autotrophic cells. Changes in the levels of triose phosphate dehydrogenase were also noted. The total pigment content of the cell and the photosynthetic electron transport reactions are not altered under different conditions of growth. PMID:16656335

  18. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase.

    PubMed

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5'-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase.

  19. 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.

  20. Action of nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and nicotinamide mononucleotide.

    PubMed

    Brunngraber, E F; Chargaff, E

    1977-10-01

    The action of the nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and on its 5'-phosphate results in the addition of one phosphate moiety to each of the substrates. Although the proof is not conclusive, it is likely that the phosphate group is transferred to the 3'-hydroxyl of the ribose. This is in contrast to the behavior of the enzyme toward NAD in which only the adenylic acid portion is phosphorylated enzymically.

  1. Sulfonate derived phosphoramidates as active intermediates in the enzymatic primer-extension of DNA.

    PubMed

    De, S; Groaz, E; Margamuljana, L; Abramov, M; Marlière, P; Herdewijn, P

    2015-04-07

    Novel unnatural 5'-phosphoramidate nucleosides, capable of being processed as substrates by DNA polymerases for multiple nucleotide incorporations, have been designed. The mimics feature metabolites such as taurine and a broad range of aliphatic sulfonates coupled through a P-N bond to the 5'-phosphate position of deoxynucleotides, to allow binding interactions in the enzyme active site. The utility of all of the analogues as pyrophosphate mimics was demonstrated for the chain elongation of DNA, using both thermophilic and mesophilic microbial polymerases.

  2. Verification, Dosimetry and Biomonitoring of Mustard Gas Exposure via Immunochemical Detection of Mustard Gas Adducts to DNA and Proteins

    DTIC Science & Technology

    1991-12-01

    of supernatants of hybridomas fox specific antibody activity . Mono- and di-adducts at the N7-position of guanosine-5-phosphate were svthesized for use...antibody activity could be developed and optimized, in which single-stranded calf-thymus DNA exposed to 10 pM mustard gas was used as coating...Figure 11: Chemical shift isuignments and coupling constants for the hydrogen (400 MHz:. a) and carbon atoms (100.6 MHz; b) of t4-(2

  3. Mutation of rpiA in Enterobacter cloacae decreases seed and root colonization and biocontrol of damping-off caused by Pythium ultimum on cucumber.

    PubMed

    Lohrke, Scott M; Dery, Pierre D; Li, Wei; Reedy, Ralph; Kobayashi, Donald Y; Roberts, Daniel R

    2002-08-01

    Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.

  4. Pnp gene modification for improved xylose utilization in Zymomonas

    SciTech Connect

    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.

  5. Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study.

    PubMed

    Shahraki, Omolbanin; Edraki, Najmeh; Khoshneviszadeh, Mehdi; Zargari, Farshid; Ranjbar, Sara; Saso, Luciano; Firuzi, Omidreza; Miri, Ramin

    2017-01-01

    Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C4 and various carboxamide substituents at C3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells' sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C4 position and compounds with 4-chlorophenyl carboxamide at C3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 µM, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 µM, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal.

  6. Hand bone loss in early rheumatoid arthritis during a methotrexate-based treat-to-target strategy with or without adalimumab-a substudy of the optimized treatment algorithm in early RA (OPERA) trial.

    PubMed

    Ørnbjerg, L M; Østergaard, M; Jensen, T; Hørslev-Petersen, K; Stengaard-Pedersen, K; Junker, P; Ellingsen, T; Ahlquist, P; Lindegaard, H; Linauskas, A; Schlemmer, A; Dam, M Y; Hansen, I; Lottenburger, T; Ammitzbøll, C G; Jørgensen, A; Krintel, S B; Raun, J; Hetland, M L; Slot, Ole; Nielsen, Lars Kjær; Skjødt, Henrik; Majgaard, Ole; Lorenzen, Tove; Horn, Hans Christian; Kowalski, Marcin; Johansen, Inger Lauge; Pedersen, Peter Mosborg; Manilo, Natalia; Bliddal, Henning

    2017-04-01

    This study aims to investigate 1-year hand bone loss (HBL1-year) in early rheumatoid arthritis (RA) patients treated with a methotrexate (MTX) and intra-articular triamcinolone treat-to-target strategy +/- adalimumab and to determine if HBL6months is associated with radiographic progression after 2 years. In a clinical trial (OPERA) of 180 treatment-naive early RA patients, bone mineral density (BMD) was estimated from hand radiographs with digital X-ray radiogrammetry (DXR) at baseline, after 6 (n = 90) and 12 months (n = 70) of follow-up. Baseline and 2-year radiographs were scored according to the Sharp/van der Heijde method. Baseline characteristics and HBL6months (0-6 months changes in DXR-BMD) were investigated as predictors of structural damage by univariate linear (∆ total Sharp/van der Heijde score (TSS) as dependent variable) and logistic (+/-radiographic progression (∆TSS >0) as dependent variable) regression analyses. Variables with p < 0.10 were included in multivariable models. In 70 patients with available HBL1-year data, HBL1-year was median (interquartile range (IQR)) -1.9 (-3.3; -0.26 mg/cm(2)) in the MTX + placebo group and -1.8 (-3.6; 0.06) mg/cm(2) in the MTX + adalimumab group, p = 0.98, Wilcoxon signed-rank. Increased HBL (compared to general population reference values) was found in 26/37 and 23/33 patients in the MTX + placebo and MTX + adalimumab groups, chi-squared = 0.99. In 90 patients with HBL6months data and 2-year radiographic data, HBL6months was independently associated with ∆TSS after 2 years (β = -0.086 (95% confidence interval = -0.15; -0.025) TSS unit/mg/cm(2) increase, p = 0.006) but not with presence of radiographic progression (∆TSS >0) (OR 0.96 (0.92-1.0), p = 0.10). In early RA patients treated with a methotrexate-based treat-to-target strategy, the majority of patients had increased HBL1-year, irrespective of adalimumab; HBL6months was independently associated with ∆TSS after 2 years.

  7. Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

    PubMed Central

    Shahraki, Omolbanin; Edraki, Najmeh; Khoshneviszadeh, Mehdi; Zargari, Farshid; Ranjbar, Sara; Saso, Luciano; Firuzi, Omidreza; Miri, Ramin

    2017-01-01

    Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C4 and various carboxamide substituents at C3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells’ sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C4 position and compounds with 4-chlorophenyl carboxamide at C3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 µM, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 µM, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal. PMID:28243063

  8. 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.

  9. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    PubMed

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  10. Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine.

    PubMed

    Commichau, Fabian M; Alzinger, Ariane; Sande, Rafael; Bretzel, Werner; Meyer, Frederik M; Chevreux, Bastien; Wyss, Markus; Hohmann, Hans-Peter; Prágai, Zoltán

    2014-09-01

    Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5'-phosphates. Pyridoxal 5'-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5'-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.

  11. Bovine brain low Mr acid phosphatase: purification and properties.

    PubMed

    Saeed, A; Tremori, E; Manao, G; Camici, G; Cappugi, G; Ramponi, G

    1990-01-01

    Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.

  12. 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.

  13. The Arabidopsis salt overly sensitive 4 Mutants Uncover a Critical Role for Vitamin B6 in Plant Salt Tolerance

    PubMed Central

    Shi, Huazhong; Xiong, Liming; Stevenson, Becky; Lu, Tiegang; Zhu, Jian-Kang

    2002-01-01

    Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis. We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene. sos4 mutant plants are hypersensitive to Na+, K+, and Li+ ions. Under NaCl stress, sos4 plants accumulate more Na+ and retain less K+ compared with wild-type plants. SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses. Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels. Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na+ and K+ homeostasis in plants. We propose that pyridoxal-5-phosphate regulates Na+ and K+ homeostasis by modulating the activities of ion transporters. PMID:11910005

  14. Branched-chain amino acid metabolon: interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm).

    PubMed

    Islam, Mohammad Mainul; Nautiyal, Manisha; Wynn, R Max; Mobley, James A; Chuang, David T; Hutson, Susan M

    2010-01-01

    The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

  15. Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.

    PubMed Central

    Hawkins, P T; Michell, R H; Kirk, C J

    1984-01-01

    We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism. PMID:6326746

  16. Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli.

    PubMed

    Lee, Pyung Cheon; Mijts, Benjamin N; Schmidt-Dannert, Claudia

    2004-10-01

    Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28 degrees C showed better production of torulene than 37 degrees C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2xYT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and beta-carotene, in E. coli. In contrast, glycerol-containing LB, 2xYT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1-3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.

  17. Enhanced production of coenzyme Q10 by self-regulating the engineered MEP pathway in Rhodobacter sphaeroides.

    PubMed

    Lu, Wenqiang; Ye, Lidan; Xu, Haoming; Xie, Wenping; Gu, Jiali; Yu, Hongwei

    2014-04-01

    Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10 ) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild-type (48.25 mg/L, 3.24 mg/g DCW).

  18. Assessment of the carcinogenic potential of high intense-sweeteners through the test for detection of epithelial tumor clones (warts) in Drosophila melanogaster.

    PubMed

    Vasconcelos, Mirley Alves; Orsolin, Priscila Capelari; Silva-Oliveira, Rosiane Gomes; Nepomuceno, Júlio César; Spanó, Mário Antônio

    2017-03-01

    High intensity-sweeteners (HIS) are natural or synthetic substances, sweeter than sugar, providing sweetness without calories. Sweeteners are mainly used as an aid in losing weight, preventing obesity and controlling blood sugar levels for diabetics. The objective of this study was to evaluate the carcinogenic potential of the sweeteners aspartame, sucralose, sodium saccharin and steviol glycoside, using the test for detection of epithelial tumor clones in Drosophila melanogaster. Larvae of 72 ± 4h, obtained from wts/TM3 female mated with mwh/mwh males, were treated for approximately 48h with different concentrations of aspartame (0.85, 1.7, 3.4, 6.8 or 13.6 mM ); sucralose (0.5, 1.25, 2.5, 5.0 or 10 mM); sodium saccharin (25; 50; 100; 200 or 400 mM) and steviol glycoside (2.5; 5.0; 10; 20 or 40 mM). Water (Reverse Osmosis) and doxorubicin (DXR 0.4 mM) were used as negative and positive controls, respectively. No statistically significant differences were observed (p > 0.05) in tumor frequencies in individuals treated with all concentrations of these sweeteners when compared to negative control. It was therefore concluded that, in these experimental conditions, aspartame, sucralose, sodium saccharin and steviol glycoside have no carcinogenic effect in D. melanogaster.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

    PubMed

    Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

    2015-02-01

    Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis.

  6. Targeting of mitochondrial Saccharomyces cerevisiae Ilv5p to the cytosol and its effect on vicinal diketone formation in brewing.

    PubMed

    Omura, Fumihiko

    2008-03-01

    Vicinal diketones (VDK) cause butter-like off-flavors in beer and are formed by a non-enzymatic oxidative decarboxylation of alpha-aceto-alpha-hydroxybutyrate and alpha-acetolactate, which are intermediates in isoleucine and valine biosynthesis taking place in the mitochondria. On the assumption that part of alpha-acetolactate can be formed also in the cytosol due to a mislocalization of the responsible acetohydroxyacid synthase encoded by ILV2 and ILV6, functional expression in the cytosol of acetohydroxyacid reductoisomerase (Ilv5p) was explored. Using the cytosolic Ilv5p, I aimed to metabolize the cytosolically formed alpha-aetolactate, thereby lowering the total VDK production. Among mutant Ilv5p enzymes with varying degrees of N-terminal truncation, one with a 46-residue deletion (Ilv5pDelta46) exhibited an unequivocal localization in the cytosol judged from microscopy of the Ilv5pDelta46-green fluorescent protein fusion protein and the inability of Ilv5pDelta46 to remedy the isoleucine/valine requirement of an ilv5Delta strain. When introduced into an industrial lager brewing strain, a robust expression of Ilv5pDelta46 was as effective as that of a wild-type Ilv5p in lowering the total VDK production in a 2-l scale fermentation trial. Unlike the case of the wild-type Ilv5p, an additional expression of Ilv5pDelta46 did not alter the quality of the resultant beer in terms of contents of aromatic compounds and organic acids.

  7. An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

    PubMed Central

    Zelenaya-Troitskaya, O; Perlman, P S; Butow, R A

    1995-01-01

    The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA. Images PMID:7621838

  8. Deletion of the gene Pip4k2c, a novel phosphatidylinositol kinase, results in hyperactivation of the immune system

    PubMed Central

    Shim, Hyeseok; Wu, Chuan; Ramsamooj, Shivan; Bosch, Kaitlyn N.; Chen, Zuojia; Emerling, Brooke M.; Yun, Jihye; Liu, Hui; Choo-Wing, Rayman; Yang, Zhiwei; Wulf, Gerburg M.; Kuchroo, Vijay Kumar; Cantley, Lewis C.

    2016-01-01

    Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kβ, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c−/− mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c−/− mice. Pip4k2c−/− mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c−/− mice and treating Pip4k2c−/− mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling. PMID:27313209

  9. De novo purine biosynthesis by two pathways in Burkitt lymphoma cells and in human spleen.

    PubMed

    Reem, G H

    1972-05-01

    This study was designed to answer the question whether human lymphocytes and spleen cells were capable of de novo purine biosynthesis. Experiments were carried out in cell-free extracts prepared from human spleen, and from a cell line established from Burkitt lymphoma. Burkitt lymphoma cells and human spleen cells could synthesize the first and second intermediates of the purine biosynthetic pathway. Cell-free extracts of all cell lines studied contained the enzyme systems which catalyze the synthesis of phosphoribosyl-1-amine, the first intermediate unique to the purine biosynthetic pathway and of phosphoribosyl glycinamide, the second intermediate of this pathway. Phosphoribosyl-1-amine could be synthesized in cell-free extracts from alpha-5-phosphoribosyl-1-pyrophosphate (PRPP) and glutamine, from PRPP and ammonia, and by an alternative pathway, directly from ribose-5-phosphate and ammonia. These findings suggest that extrahepatic tissues may be an important source for the de novo synthesis of purine ribonucleotide in man. They also indicate that ammonia may play an important role in purine biosynthesis. The alternative pathway for the synthesis of phosphoribosyl-1-amine from ribose-5-phosphate and ammonia was found to be subject to inhibition by the end products of the purine synthetic pathway, particularly by adenylic acid and to a lesser degree by guanylic acid. The alternative pathway for phosphoribosyl-1-amine synthesis from ribose-5-phosphate and ammonia may contribute significantly towards the regulation of the rate of de novo purine biosynthesis in the normal state, in metabolic disorders in which purines are excessively produced and in myeloproliferative diseases.

  10. Uptake of N-(4'-pyridoxyl)amines and release of amines by renal cells: a model for transporter-enhanced delivery of bioactive compounds.

    PubMed Central

    Zhang, Z M; McCormick, D B

    1991-01-01

    The importing of vitamin B6 by renal proximal tubular cells from the rat is facilitated and Na(+)-dependent and reflects specificity for the meta-phenolate pyridinium structure with a 5-hydroxymethyl function. This transporter can, however, accept competitively each of the natural nonphosphorylated vitamers (pyridoxine, pyridoxamine, and pyridoxal) and other B6 analogues differing only in the groups at position 4. A series of N-(4'-pyridoxyl)amines was synthesized by sodium borohydride or boro[3H]hydride reduction of aldimines formed by condensing the amines with pyridoxal. The unlabeled B6-secondary amine compounds were found to competitively inhibit the uptake of [4'-3H]pyridoxine by the renal cells. Moreover, the 3H-labeled N-(4'-pyridoxyl)amines were shown to enter the cells by the process facilitated by the B6 transporter. Upon entry the labeled compounds were converted to N-(5'-phospho-4'-pyridoxyl)amines in a reaction catalyzed by pyridoxal kinase, an enzyme that tolerates considerable functional variation in position 4 of the B6 structure. The 5'-phosphates were subsequently converted within the cell to pyridoxal 5'-phosphate with liberation of the original amine in a reaction catalyzed by pyridoxamine (pyridoxine) 5'-phosphate oxidase, an enzyme with broad specificity for 4'-substituted amines on the 5'-phospho-B6 structure. This system illustrates how knowledge of transporter specificity can permit design of a compound with potential biologic activity. A drug or other intracellular effector may be piggybacked onto a transported solute (e.g., vitamin or other nutrient) that gains facilitated entry to a cell and is, thereafter, metabolized to release the active compound. PMID:1961706

  11. 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

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

    PubMed

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

    2010-04-01

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

  13. Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

    PubMed

    Smith, Amber Marie; Brown, William Clay; Harms, Etti; Smith, Janet L

    2015-02-27

    PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.

  14. Crystal Structure of Pyridoxal Biosynthesis Lyase PdxS from Pyrococcus horikoshii

    PubMed Central

    Matsuura, Atsushi; Yoon, Ji Young; Yoon, Hye-Jin; Lee, Hyung Ho; Suh, Se Won

    2012-01-01

    Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6 and is de novo synthesized from three substrates, dihydroxyacetone phosphate (DHAP), riburose 5-phosphate (RBP), and ammonia hydrolysed from glutamine. Glutamine amidotransferase (PdxT) catalyzes the production of ammonia from glutamine, while PdxS catalyzes the following condensation of ribulose 5-phosphate (Ru5P), glyceraldehyde-3-phosphate (G3P), and ammonia. PdxS exists as a hexamer or dodecamer depending on species and makes a 1:1 complex with PdxT. Pyrococcus horikoshii PdxS has a 37 amino acids insertion region, which is found in some archaeal PdxS proteins, but its structure and function are unknown. To provide further structural information on the role of the insertion region, the oligomeric state, and ligand binding mode of P. horikoshii PdxS, the crystal structure of PdxS from P. horikoshii was solved in two forms: (i) apo form, (ii) r ibose 5-phosphate (R5P) complex and the quaternary structure of PdxS in solution was determined by analytical gel filtration. P. horikoshii PdxS forms hexamer in solution based on analytical gel filtration data. When we superimpose the structure of P. horikoshii PdxS with other dodecamer structures of PdxS, the additional insertion is located apart from the active site and induces a steric clash on the hexamer-hexamer interface of PdxS proteins. Our results suggest that the additional insertion perturbs dodecamer formation of P. horikoshii PdxS. PMID:23104439

  15. 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

  16. 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.

  17. Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction.

    PubMed

    Zaug, A J; Grabowski, P J; Cech, T R

    The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.

  18. 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

    Mills, Stephen J; Silvander, Camilla; Cozier, Gyles; Trésaugues, Lionel; Nordlund, Pär; Potter, Barry V L

    2016-03-08

    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.

  19. Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

    PubMed

    Kikuchi, Y; Tyc, K; Filipowicz, W; Sänger, H L; Gross, H J

    1982-12-11

    A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.

  20. 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

  1. The chicken B-cell line DT40 proteome, beadome and interactomes

    PubMed Central

    Rees, Johanna S.; Lilley, Kathryn S.; Jackson, Antony P.

    2015-01-01

    In developing a new quantitative AP-MS method for exploring interactomes in the chicken B-cell line DT40, we also surveyed the most abundant proteins in this organism and explored the likely contaminants that bind to a variety of affinity resins that would later be confirmed quantitatively [1]. We present the ‘Top 150 abundant DT40 proteins list’, the DT40 beadomes as well as protein interaction lists for the Phosphatidyl inositol 5-phosphate 4-kinase 2β and Fanconi anaemia protein complexes. PMID:26217713

  2. Aminoacyl-tRNA analogues; synthesis, purification and properties of 3'-anthraniloyl oligoribonucleotides.

    PubMed

    Nawrot, B; Sprinzl, M

    1998-04-01

    Reaction of isatoic anhydride with adenosine, adenosine 5'-phosphate, oligoribonucleotides or with the E. coli tRNAVal led to attachment of an anthraniloyl residue at 2'- or 3'-OH groups of 3'-terminal ribose residue. No protection of the 5'-hydroxyl group or internal 2'-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

  3. Efficient application of monolithic silica column to determination of illicit heroin street sample by HPLC.

    PubMed

    Macchia, Marco; Bertini, Simone; Mori, Claudio; Orlando, Caterina; Papi, Chiara; Placanica, Giorgio

    2004-03-01

    In this paper, an HPLC method is proposed for a routine, rapid and simple analysis of heroin samples confiscated from the illicit market, based on a new type of packing for HPLC columns (monolithic silica). Acetonitrile and pH 3.5 phosphate buffer solution were used under both isocratic and gradient conditions. Under our analytical conditions, all the components of a typical mixture of an illicit heroin sample proved to be fully separated into well-resolved peaks in 7 min. Analytical linearity and accuracy of the method were also studied for all analytes using tetracaine hydrochloride as the internal standard.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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 +.

  11. 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.

  12. Responses of Cholinergic and Noncholinergic Renshaw Cell Receptors After Acute and Chronic Exposure to Anticholinesterases.

    DTIC Science & Technology

    1983-07-01

    observed for symptoms of toxicity. 5." 9..-,,,, ,,. < 7- -. Prior to removal of tissue samples, cats were anesthetized with DIAL (80mg/kg, i.p...cord was transected at LI following administration of 2% lidocaine. Tissue samples (dorsal and ventral spinal roots as well as 2-3mm sections of the...investigation of ventral horn interneurons. The tissue samples were fixed by immersion in 5% phosphate buffered (0.1M, pH 7.4) glutaraldehyde. After 24

  13. 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

  14. 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

  15. Modulation of P-glycoprotein-mediated multidrug resistance in K562 leukemic cells by indole-3-carbinol

    SciTech Connect

    Arora, Annu; Seth, Kavita; Kalra, Neetu; Shukla, Yogeshwer . E-mail: yogeshwer_shukla@hotmail.com

    2005-02-01

    Resistance to chemotherapeutic drugs is one of the major problems in the treatment of cancer. P-glycoprotein (P-gp) encoded by the mdr gene is a highly conserved protein, acts as a multidrug transporter, and has a major role in multiple drug resistance (MDR). Targeting of P-gp by naturally occurring compounds is an effective strategy to overcome MDR. Indole-3-carbinol (I3C), a glucosinolates present in cruciferous vegetables, is a promising chemopreventive agent as it is reported to possess antimutagenic, antitumorigenic, and antiestrogenic properties in experimental studies. In the present investigation, the potential of I3C to modulate P-gp expression was evaluated in vinblastine (VBL)-resistant K562 human leukemic cells. The resistant K562 cells (K562/R10) were found to be cross-resistant to vincristine (VCR), doxorubicin (DXR), and other antineoplastic agents. I3C at a nontoxic dose (10 x 10{sup -3} M) enhanced the cytotoxic effects of VBL time dependently in VBL-resistant human leukemia (K562/R10) cells but had no effect on parent-sensitive cells (K562/S). The Western blot analysis of K 562/R 10 cells showed that I3C downregulates the induced levels of P-gp in resistant cells near to normal levels. The quantitation of immunocytochemically stained K562/R10 cells showed 24%, 48%, and 80% decrease in the levels of P-gp by I3C for 24, 48, and 72 h of incubation. The above features thus indicate that I3C could be used as a novel modulator of P-gp-mediated multidrug resistance in vitro and may be effective as a dietary adjuvant in the treatment of MDR cancers.

  16. Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor.

    PubMed

    Ahlawat, Seema; Saxena, Parul; Ali, Athar; Khan, Shazia; Abdin, Malik Z

    2017-02-17

    Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways.

  17. Predicting the failure load of the distal radius.

    PubMed

    Muller, Monique E; Webber, Colin E; Bouxsein, Mary L

    2003-06-01

    The distal radius is an important site for the early detection of patients at risk for fracture. Since measuring bone strength in vivo is not possible, we evaluated which bone assessment method of the forearm would best predict failure load of the distal radius and computed a factor of risk for wrist fracture (Phi wrist). Thirty-eight cadaveric forearm specimens were measured by five different techniques to assess bone density, bone mineral content, geometry and trabecular structure at the distal forearm. The bone assessment techniques included dual-energy X-ray absorptiometry (DXA) of the radius, peripheral quantitative computed tomography (pQCT) of the 4% and 20% distal sites of the radius, DXA of the phalanges, digital X-ray radiogrammetry of the forearm (DXR-BMD), and quantitative ultrasound of the radius. The failure load of each excised radius was determined by simulating a fall on an outstretched hand. The pQCT measurements of polar stress-strain index and cortical content explained the greatest portion of variance in failure load (r2=0.82-0.85). Bone mineral content measures were generally better predictors of failure load (r2=0.53-0.85) than the corresponding volumetric or areal bone mineral density values (r2=0.22-0.69) measured by either pQCT or DXA. Multiple regression analysis showed that the addition of a bone geometry measure improved the ability of a bone density measure alone to predict failure load. There was high variability in the ability of different techniques and different variables within a given technique to predict failure load. Estimates of the factor of risk for wrist fracture (Phi wrist) revealed that the women in this study would have been likely to fracture their distal radius upon falling from a standing height (Phi wrist= 1.04), whereas the men would have likely withstood the impact without fracturing their wrist (Phi wrist= 0.79).

  18. 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.

  19. Elevation of night-time temperature increases terpenoid emissions from Betula pendula and Populus tremula

    PubMed Central

    Ibrahim, Mohamed A.; Mäenpää, Maarit; Hassinen, Viivi; Kontunen-Soppela, Sari; Malec, Lukáš; Rousi, Matti; Pietikäinen, Liisa; Tervahauta, Arja; Kärenlampi, Sirpa; Holopainen, Jarmo K.; Oksanen, Elina J.

    2010-01-01

    Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 °C) while daytime temperature was kept at a constant 22 °C. VOC emissions were collected during the daytime and analysed by gas chromatography–mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and β-ocimene increased from 6 °C to 14 °C, while several other monoterpenes and the SQTs (Z,E)-α-farnesene and (E,E)-α-farnesene increased up to 18 °C. Total monoterpene and sesquiterpene emission peaked at 18 °C, whereas isoprene emissions decreased at 22 °C. Leaf area increased across the temperature range of 6–22 °C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees. PMID:20181662

  20. 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.

  1. 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

  2. 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.

  3. LytB1 and LytB2 of Mycobacterium tuberculosis Are Not Genetically Redundant

    PubMed Central

    Brown, Amanda Claire; Kokoczka, Rachel; Parish, Tanya

    2015-01-01

    Mycobacterium tuberculosis synthesises isoprenoid precursors via the MEP/DOXP pathway and at least five enzymes in the pathway (Dxs1, Dxr/IspC, IspD, IspF, and GcpE/IspG) are required for growth in vitro. We investigated the role of LytB (IspH) in M. tuberculosis; M. tuberculosis is unusual in that it has two homologs–LytB1 and LytB2. We were unable to delete the lytB2 gene unless we provided an additional copy elsewhere, demonstrating that this is the essential homolog. We expressed lytB1 from the lytB2 promoter and confirmed that this could not complement for loss of function of lytB2, despite LytB1 possessing all the previously described conserved critical residues. Interestingly the sole LytB homolog of Mycobacterium smegmatis was able to compensate for loss of LytB2 in M. tuberculosis. We tested translational fusions of LytB1 and LytB2 for functionality in M. tuberculosis, but only a fusion with 90% N-terminal LytB2 and 10% C-terminal LytB1 was functional. In order to identify the key difference between the two proteins, site directed mutagenesis was used to change LytB2 residues into their counterparts in LytB1. None of these amino acid substitutions was essential for function and all lytB2 mutant alleles were functional. In contrast, mutation of the key residues for [Fe4S4] cluster formation, as well as a catalytic residue in LytB1 did not result in functional complementation. Thus, although LytB1 and LytB2 are not genetically redundant, this is not dependent on small amino acid changes, but is likely to be a result of major overall structural differences. PMID:26309039

  4. Land snails as a diet diversification proxy during the early upper palaeolithic in Europe.

    PubMed

    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.

  5. Elevation of night-time temperature increases terpenoid emissions from Betula pendula and Populus tremula.

    PubMed

    Ibrahim, Mohamed A; Mäenpää, Maarit; Hassinen, Viivi; Kontunen-Soppela, Sari; Malec, Lukás; Rousi, Matti; Pietikäinen, Liisa; Tervahauta, Arja; Kärenlampi, Sirpa; Holopainen, Jarmo K; Oksanen, Elina J

    2010-06-01

    Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 degrees C) while daytime temperature was kept at a constant 22 degrees C. VOC emissions were collected during the daytime and analysed by gas chromatography-mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and beta-ocimene increased from 6 degrees C to 14 degrees C, while several other monoterpenes and the SQTs (Z,E)-alpha-farnesene and (E,E)-alpha-farnesene increased up to 18 degrees C. Total monoterpene and sesquiterpene emission peaked at 18 degrees C, whereas isoprene emissions decreased at 22 degrees C. Leaf area increased across the temperature range of 6-22 degrees C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees.

  6. Antisense sequence-directed cross-linking of DNA oligonucleotides by mitomycin C.

    PubMed

    Maruenda, H; Tomasz, M

    1996-01-01

    Oligodeoxyribonucleotides (ODNs) conjugated with mitomycin C (MC) via (-CH2-)n tethers of different lengths (n = 6, 12) to their terminal 5'-phosphate were synthesized, and their interaction with target complementary single-stranded DNA oligonucleotides was investigated. MC, a clinically used natural anticancer drug, is known to act as a bioreductive alkylating agent of duplex DNA with a remarkable preference for 5'-d(CG) sequences. The usual enzymatic bioreductive techniques known to trigger MC to alkylate DNA were employed in the reaction between the MC-oligonucleotide conjugates and their targets radiolabeled by 32P at their 5'-phosphate. A slow-moving radiolabeled product, detected by polyacrylamide gel electrophoresis using phosphorimaging techniques, was obtained in 15-25% yield with complementary DNA as target. Formation of these products was dependent upon complementary duplex formation. Evidence is presented that the DNA target is alkylated by the mitomycin C moiety of the ODN conjugate at the 2-amino group of a guanine base. These findings suggest that the MC-ODN conjugates may be useful specific inhibitors of cellular or viral gene expression. To our knowledge this is the first report on ODN conjugates of a reductively activated drug of known therapeutic value.

  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. Anion and divalent cation activation of phosphoglycolate phosphatase from leaves.

    PubMed

    Husic, H D; Tolbert, N E

    1984-02-15

    Phosphoglycolate (P-glycolate) phosphatase was purified 223-fold from spinach leaves by (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and Sephadex G-200 chromatography. The partially purified enzyme had a broad pH optimum between 5.6 and 8.0 and was specific for the hydrolysis of P-glycolate with a Km (P-glycolate) of 26 microM. The enzyme was activated by divalent cations including Mg2+, Co2+, Mn2+, and Zn2+, and by anions including Cl-, Br-, NO-3, and HCOO-. Neither anions nor divalent cations activated the enzyme without the other. The P-glycolate phosphatase activities from tobacco leaves or the green algae, Chlamydomonas reinhardtii, also required Mg2+ and were activated by chloride. In addition, the enzyme was allosterically inhibited by ribose 5-phosphate. The activation of P-glycolate phosphatase by both anions and divalent cations and the inhibition by ribose 5-phosphate may be involved in the in vivo regulation of P-glycolate phosphatase activity.

  9. Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

    PubMed

    Rodrigues, Matthew J; Windeisen, Volker; Zhang, Yang; Guédez, Gabriela; Weber, Stefan; Strohmeier, Marco; Hanes, Jeremiah W; Royant, Antoine; Evans, Gwyndaf; Sinning, Irmgard; Ealick, Steven E; Begley, Tadhg P; Tews, Ivo

    2017-03-01

    Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand β6 of the Pdx1 (βα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

  10. The L-arabinose-resistance test with Salmonella typhimurium strain SV3 selects forward mutations at several ara genes.

    PubMed

    Pueyo, C; Lopez-Barea, J

    1979-08-01

    A new assay has been described for mutagenicity testing using an L-arabinose-sensitive strain of Salmonella typhimurium. The test strain SV3 and several L-arabinose-resistant mutants selected therefrom are characterized in the present study by 3 different criteria: inhibition of growth by L-arabinose, accumulation of keto-sugars, and activities of the enzymes involved in L-arabinose catabolism. Strain SV3 (ara-531) shows high levels of inducible L-arabinose isomerase (EC 5.3.1.4) and L-ribulokinase (EC 2.7.1.16) activities, but is deficient in L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4), the enzyme encoded in Escherichia coli by gene D in the araBAD operon. Addition of L-arabinose to SV3 growing in glycerol or casamino acids stops growth. D-Glucose only partially reverses this inhibition. Reversion of the ara-531 mutation restores different levels of epimerase activity and resistance to L-arabinose. However, the great majority of the L-arabinose-resistant mutants do not utilize L-arabinose. The physiological and enzymatic properties of these L-arabinose non-utilizing mutants suggest that L-arabinose resistance is due to forward mutations in at least 3 other genes, araA, araB and araC, blocking steps prior to L-ribulose 5-phosphate accumulation.

  11. Calvin cycle multienzyme complexes are bound to chloroplast thylakoid membranes of higher plants in situ.

    PubMed Central

    Suss, K H; Arkona, C; Manteuffel, R; Adler, K

    1993-01-01

    Further evidence is provided that the Calvin cycle enzymes ribose-5-phosphate isomerase (EC 5.3.1.6), ribulose-5-phosphate kinase (Ru-5-P-K, EC 2.7.1.19), ribulose-1,5-bisphosphate carboxylase (RuP2Case, EC 4.1.1.39), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), sedoheptulose-1,7-bisphosphatase (Sed-1,7-bPase, EC 3.1.3.37), and electron transport protein ferredoxin-NADP+ reductase (FNR, EC 1.18.1.1) are organized into stable CO2-fixing multienzyme complexes with a molecular mass of 900 kDa. Limited trypsinolysis combined with immunoblotting revealed that all of chloroplast stromal Ru-5-P-K and GAPDH is located in enzyme complexes. The Calvin cycle enzyme complexes remain intact indefinitely at lower ionic strength but dissociate into components at KCl concentrations >250 mM. Immunoelectron microscopy showed that Ru-5-P-K, GAPDH, Sed-1,7-bPase, and FNR are bound to stroma-faced thylakoid membranes in situ, whereas RuP2Case and RuP2Case activase are randomly distributed throughout chloroplasts. The results indicate that membrane-bound enzyme supercomplexes may play an important role in photosynthesis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:11607406

  12. Impact of Pre-Pregnancy BMI on B Vitamin and Inflammatory Status in Early Pregnancy: An Observational Cohort Study.

    PubMed

    Bjørke-Monsen, Anne-Lise; Ulvik, Arve; Nilsen, Roy M; Midttun, Øivind; Roth, Christine; Magnus, Per; Stoltenberg, Camilla; Vollset, Stein Emil; Reichborn-Kjennerud, Ted; Ueland, Per Magne

    2016-11-30

    Maternal nutrition and inflammation have been suggested as mediators in the development of various adverse pregnancy outcomes associated with maternal obesity. We have investigated the relation between pre-pregnancy BMI, B vitamin status, and inflammatory markers in a group of healthy pregnant women. Cobalamin, folate, pyridoxal 5'-phosphate, and riboflavin; and the metabolic markers homocysteine, methylmalonic acid, and 3-hydroxykynurenine/xanthurenic acid ratio (HK/XA); and markers of cellular inflammation, neopterin and kynurenine/tryptophan ratio (KTR) were determined in pregnancy week 18 and related to pre-pregnancy body mass index (BMI), in 2797 women from the Norwegian Mother and Child Cohort Study (MoBa). Pre-pregnancy BMI was inversely related to folate, cobalamin, pyridoxal 5'-phosphate (PLP), and riboflavin (p < 0.001), and associated with increased neopterin and KTR levels (p < 0.001). Inflammation seemed to be an independent predictor of low vitamin B6 status, as verified by low PLP and high HK/XA ratio. A high pre-pregnancy BMI is a risk factor for low B vitamin status and increased cellular inflammation. As an optimal micronutrient status is vital for normal fetal development, the observed lower B vitamin levels may contribute to adverse pregnancy outcomes associated with maternal obesity and B vitamin status should be assessed in women with high BMI before they get pregnant.

  13. Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment

    SciTech Connect

    Tsuchiya, Hiroyuki; Harashima, Hideyoshi; Kamiya, Hiroyuki . E-mail: hirokam@pharm.hokudai.ac.jp

    2005-11-04

    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment ({approx}600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

  14. An Adaptation To Life In Acid Through A Novel Mevalonate Pathway

    PubMed Central

    Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.; Bowie, James U.

    2016-01-01

    Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments. PMID:28004831

  15. 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.

  16. Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

    PubMed

    Suzuki, Ryuichiro; Katayama, Takane; Kim, Byung-Jun; Wakagi, Takayoshi; Shoun, Hirofumi; Ashida, Hisashi; Yamamoto, Kenji; Fushinobu, Shinya

    2010-10-29

    Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

  17. 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.

  18. DFT studies on Schiff base formation of vitamin B6 analogues. Reaction between a pyridoxamine-analogue and carbonyl compounds.

    PubMed

    Ortega-Castro, J; Adrover, M; Frau, J; Salvà, A; Donoso, J; Muñoz, F

    2010-04-08

    A comprehensive theoretical study based on density functional theory calculations (B3LYP and M06-2X functionals) of the formation of Schiff bases of pyridoxamine analogues with two different aldehydes was conducted. The reaction mechanism was found to involve two steps, namely: (1) formation of a carbinolamine and (2) dehydration of the carbinolamine to give the final imine. Also, consistent with available experimental evidence, the carbinolamine dehydration was the rate-determining step of the process determined by means of M06-2X functional. Using an appropriate solvation method and reactant conformation ensures that all proton transfers involved will be intramolecular, which substantially reduces energy barriers and facilitates reaction in all cases. The formation of a Schiff base between pyridoxal 5-phosphate (PLP) and an amine or amino acid requires the contribution of an external water molecule in order to facilitate proton transfers. On the other hand, the formation of a Schiff base between pyridoxamine 5-phosphate (PMP) and a carbonyl compound requires no external aid since the spatial arrangement of the functional groups in PMP ensures that all proton transfers will be intramolecular.

  19. Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat.

    PubMed Central

    Stipanuk, M H; Beck, P W

    1982-01-01

    The contribution of cystathionine gamma-lyase, cystathionine beta-synthase and cysteine aminotransferase coupled to 3-mercaptopyruvate sulphurtransferase to cysteine desulphhydration in rat liver and kidney was assessed with four different assay systems. Cystathionine gamma-lyase and cystathionine beta-synthase were active when homogenates were incubated with 280 mM-L-cysteine and 3 mM-pyridoxal 5'-phosphate at pH 7.8. Cysteine aminotransferase in combination with 3-mercaptopyruvate sulphurtransferase catalysed essentially all of the H2S production from cysteine at pH 9.7 with 160 mM-L-cysteine, 2 mM-pyridoxal 5'-phosphate, 3 mM-2-oxoglutarate and 3 mM-dithiothreitol. At more-physiological concentrations of cysteine (2 mM) cystathionine gamma-lyase and cystathionine beta-synthase both appeared to be active in cysteine desulphhydration, whereas the aminotransferase pathway did not. The effect of inhibition of cystathionine gamma-lyase by a suicide inactivator, propargylglycine, in the intact rat was also investigated; there was no significant effect of propargylglycine administration on the urinary excretion of total 35S, 35SO4(2-) or [35S]taurine formed from labelled dietary cysteine. PMID:7150244

  20. 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.

  1. Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana 1

    PubMed Central

    Liu, Shih-Tung; Katz, Charles D.; Knight, C. Arthur

    1978-01-01

    The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid. In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [14C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices. It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants. The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined. PMID:16660376

  2. 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.

  3. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    PubMed

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.

  4. Development of a comprehensive analytical method for phosphate metabolites in plants by ion chromatography coupled with tandem mass spectrometry.

    PubMed

    Sekiguchi, Yoko; Mitsuhashi, Naoto; Kokaji, Tetsuo; Miyakoda, Hidekazu; Mimura, Tetsuro

    2005-08-26

    This paper describes the development of a practical method for the analysis of phosphorus compounds with a focus on sugar phosphates from the model higher plant Arabidopsis thaliana by ion chromatography coupled to electrospray ionization tandem mass spectrometry (IC-ESI-MS-MS). After the analytical separation, the potassium hydroxide eluent was converted to water with an anion suppressor allowing the effluent from the IC to be connected to the mass spectrometer directly. In the optimized method, 17 phosphorous compounds (adenosine diphosphate (ADP), fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, fructose 6-phosphate, galactose 1-phosphate, glucose 1-phosphate, glucose 1,6-bisphosphate, glucose 6-phosphate, mannose 6-phosphate, phosphoenol pyrvate, 3-phosphoglyceric acid, ribulose 1,5-bisphosphate, ribulose 5-phosphate, ribose 5-phosphate, sucrose 6-phosophate and uridine 5'-diphosphate-glucose (UDPG)) were determined. The linearity of response for these phosphorous compounds over the concentration range of 0 and 10 microM was better than 0.9993 in all cases. The minimum detection limit was between 0.01 and 2.50 microM for a 25 microL injection, and recovery rates for standard addition to the sample were within the range from 93% to 110%.

  5. 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.

  6. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

    PubMed

    Kuyper, Marko; Hartog, Miranda M P; Toirkens, Maurice J; Almering, Marinka J H; Winkler, Aaron A; van Dijken, Johannes P; Pronk, Jack T

    2005-02-01

    After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

  7. 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.

  8. Factors influencing pyrroline 5-carboxylate synthesis from glutamate by rat intestinal mucosa mitochondria

    SciTech Connect

    Henslee, J.G.; Wakabayashi, Y.; Small, C.; Jones, M.E.

    1983-10-15

    Factors influencing pyrroline 5-carboxylate (P5C) synthesis from glutamate by a subcellular fraction enriched in mitochondria of rat small intestinal mucosa have been studied. P5C synthesis decreased rapidly if this subcellular fraction was preincubated at 20 degrees C in the absence of substrates; this effect suggests that the enzyme(s) catalyzing P5C synthesis from glutamate (P5C synthase) is unstable in the absence of substrates. In the presence of substrates P5C synthesis increased linearly for the first 30 min of incubation, suggesting that the substrates promote enzyme stability. Pyridoxal 5'-phosphate is an effective inhibitor of P5C synthase whereas pyridoxamine 5'-phosphate and pyridoxal are not inhibitory. Potassium phosphate, KCl, and KBr each inhibited P5C synthase but potassium-Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) did not. Potassium phosphate was the most potent inhibitor followed by KBr, and then KCl. These results suggest P5C synthase is sensitive to anion inhibition. Both L-ornithine and D-ornithine inhibited P5C synthase; L-proline did not inhibit. Several analogs of ornithine and proline were also tested and none was found to inhibit P5C synthase; the inhibition by ornithine is, therefore, rather specific and it may prove to contribute to the regulation of metabolism of these amino acids.

  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. 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

  11. 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; ...

    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

  12. Insights into the mechanism of Pseudomonas dacunhae aspartate beta-decarboxylase from rapid-scanning stopped-flow kinetics.

    PubMed

    Phillips, Robert S; Lima, Santiago; Khristoforov, Roman; Sudararaju, Bakthavatsalam

    2010-06-22

    The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate beta-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium l-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5'-phosphate and oxaloacetate or pyruvate. After consumption of the l-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate. In contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-beta-hydroxy-dl-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of approximately 1 or 0.006 s(-1), respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of l-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. It is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5'-phosphate Schiff base and increases the pK(a), resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM l-Asp also results in the formation of the 325 nm complex, but with an apparent rate constant of 0.2 s(-1), at least 5000-fold slower than the rate of wild-type ABDC. In contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role

  13. 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.

  14. Rapid determination of thiamine, riboflavin, pyridoxine, and niacinamide in infant formulas by liquid chromatography.

    PubMed

    Woollard, David C; Indyk, Harvey E

    2002-01-01

    A simplified, simultaneous determination of vitamins B1, B2, B3, and B6 in supplemented infant formulas was developed from a single deproteinized sample extract, with analysis by reversed-phase, ion-pair chromatography with an acidified methanol-water mobile phase. The dioctylsulfosuccinate counter-ion facilitates unique retention of the pyridine-based vitamins (niacinamide and pyridoxine) and allows for concurrent measurement of both the pyridoxal and riboflavin 5'-phosphate endogenous components of milk. Other naturally occurring undetected vitamin congeners have minimal analytical significance. UV detection is used for niacinamide, and programmed fluorescence detection is used for riboflavin and the B6 vitamins. Thiamine is routinely determined sequentially under modified elution conditions.

  15. Complementation of an Escherichia coli pyrF mutant with DNA from Desulfovibrio vulgaris

    SciTech Connect

    Li, C.; Peck, H.D. Jr.; Przybyla, A.E.

    1986-02-01

    A PyrF/sup -/ mutant of Escherichia coli (SK1108, pyrF::Tn5 Kan/sup r/) was complemented with the Desulfovibrio vulgaris (Hildenborough) structural gene for orotidine-5'-phosphate decarboxylase. Either orientation of a 1.6-kilobase-pair D. vulgaris DNA fragment (pLP3B or pLP3A) complemented the PyrF/sup -/ strain suggesting that the D. vulgaris pyrF promoter was functional. The apparent product of the D. vulgaris pyrF gene was a single 26-kilodalton polypeptide. These results demonstrate the utility of E. coli cloning systems in studying metabolic and energetic pathways in sulfate-reducing bacteria.

  16. 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.

  17. Structure of ALD1, a plant-specific homologue of the universal diaminopimelate aminotransferase enzyme of lysine biosynthesis.

    PubMed

    Sobolev, Vladimir; Edelman, Marvin; Dym, Orly; Unger, Tamar; Albeck, Shira; Kirma, Menny; Galili, Gad

    2013-02-01

    Diaminopimelate aminotransferase (DAP-AT) is an enzyme in the lysine-biosynthesis pathway. Conversely, ALD1, a close homologue of DAP-AT in plants, uses lysine as a substrate in vitro. Both proteins require pyridoxal-5'-phosphate (PLP) for their activity. The structure of ALD1 from the flowering plant Arabidopsis thaliana (AtALD1) was solved at a resolution of 2.3 Å. Comparison of AtALD1 with the previously solved structure of A. thaliana DAP-AT (AtDAP-AT) revealed similar interactions with PLP despite sequence differences within the PLP-binding site. However, sequence differences between the binding site of AtDAP-AT for malate, a purported mimic of substrate binding, and the corresponding site in AtALD1 led to different interactions. This suggests that either the substrate itself, or the substrate-binding mode, differs in the two proteins, supporting the known in vitro findings.

  18. Optimization of Direct Lysine Decarboxylase Biotransformation for Cadaverine Production with Whole-Cell Biocatalysts at High Lysine Concentration.

    PubMed

    Kim, Hyun Joong; Kim, Yong Hyun; Shin, Ji-Hyun; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Seo, Hyung-Min; Choi, Kwon Young; Yang, Yung-Hun; Park, Kyungmoon

    2015-07-01

    Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.

  19. 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

  20. The synthesis of 4-methylumbelliferyl phenylphosphonate and its used in an improved method for the zymogram analysis of phosphodiesterase I.

    PubMed

    Hawley, D M; Crisp, M; Hodes, M E

    1983-03-01

    The 4-methylumbelliferyl analog of p-nitrophenyl phenylphosphonate was prepared and its cleavage by different phosphohydrolases was studied. The new compound, 4-methylumbelliferyl phenylphosphonate, is hydrolyzed by purified phosphodiesterase I (EC 3.1.4.1) from snake venom or bovine intestinal mucosa. This fact has been exploited to develop a zymogram method which allows the detection of these phosphohydrolases on gels, even when the enzymes are present in small amounts in crude extracts. This method is superior to chromogenic methods evolving p-nitrophenol in that it is far more sensitive and is superior to the 4-methylumbelliferyl thymidine 5'-phosphate method in that the substrate is easier to prepare and can be isolated in large quantity.

  1. Electrochemical detection of DNA hybridization by using a zirconia modified renewable carbon paste electrode.

    PubMed

    Zuo, Shao-Hua; Zhang, Ling-Fan; Yuan, Hui-Hui; Lan, Min-Bo; Lawrance, Geoffrey A; Wei, Gang

    2009-02-01

    A simple, polishable and renewable DNA biosensor was fabricated based on a zirconia modified carbon paste electrode. Zirconia was mixed with graphite powder and paraffin wax to produce the paste for the electrode, and response-optimized at 56% graphite powder, 19% ZrO(2) and 25% paraffin wax. An oligonucleotide probe with a terminal 5'-phosphate group was attached to the surface of the electrode via the strong affinity of zirconia for phosphate groups. DNA immobilization and hybridization were characterized by cyclic voltammetry and differential pulse voltammetry, using methylene blue as indicator. Examination of changes in response with complementary or non-complementary DNA sequences showed that the developed biosensor had a high selectivity and sensitivity towards hybridization detection (< or =2x10(-10) M complementary DNA detectable). The surface of the biosensor can be renewed quickly and reproducibly (signal RSD+/-4.6% for five successive renewals) by a simple polishing step.

  2. Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

    PubMed Central

    Kikuchi, Y; Tyc, K; Filipowicz, W; Sänger, H L; Gross, H J

    1982-01-01

    A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini. Images PMID:6760127

  3. Structural basis for phosphoinositide substrate recognition, catalysis, and membrane interactions in human inositol polyphosphate 5-phosphatases.

    PubMed

    Trésaugues, Lionel; Silvander, Camilla; Flodin, Susanne; Welin, Martin; Nyman, Tomas; Gräslund, Susanne; Hammarström, Martin; Berglund, Helena; Nordlund, Pär

    2014-05-06

    SHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family.

  4. 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.

  5. Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp.

    PubMed

    Luan, Fang; Wüst, Matthias

    2002-07-01

    In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.

  6. Disruption of a Topoisomerase-DNA Cleavage Complex by a DNA Helicase

    NASA Astrophysics Data System (ADS)

    Howard, Michael T.; Neece, Sue H.; Matson, Steven W.; Kreuzer, Kenneth N.

    1994-12-01

    The type II DNA topoisomerases are targets for a variety of chemotherapeutic agents, including the antibacterial quinolones and several families of antitumor drugs. These agents stabilize an enzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drug-stabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons.

  7. Vitroprocines, new antibiotics against Acinetobacter baumannii, discovered from marine Vibrio sp. QWI-06 using mass-spectrometry-based metabolomics approach.

    PubMed

    Liaw, Chih-Chuang; Chen, Pei-Chin; Shih, Chao-Jen; Tseng, Sung-Pin; Lai, Ying-Mi; Hsu, Chi-Hsin; Dorrestein, Pieter C; Yang, Yu-Liang

    2015-08-04

    A robust and convenient research strategy integrating state-of-the-art analytical techniques is needed to efficiently discover novel compounds from marine microbial resources. In this study, we identified a series of amino-polyketide derivatives, vitroprocines A-J, from the marine bacterium Vibrio sp. QWI-06 by an integrated approach using imaging mass spectroscopy and molecular networking, as well as conventional bioactivity-guided fractionation and isolation. The structure-activity relationship of vitroprocines against Acinetobacter baumannii is proposed. In addition, feeding experiments with (13)C-labeled precursors indicated that a pyridoxal 5'-phosphate-dependent mechanism is involved in the biosynthesis of vitroprocines. Elucidation of amino-polyketide derivatives from a species of marine bacteria for the first time demonstrates the potential of this integrated metabolomics approach to uncover marine bacterial biodiversity.

  8. Location of protein S1 of Escherichia coli ribosomes at the 'A'-site of the codon binding site. Affinity labeling studies with a 3'-modified A-U-G analog.

    PubMed Central

    Pongs, O; Stöffler, G; Bald, R W

    1976-01-01

    An affinity analog with a 5-bromoacetamido uridine 5'-phosphate moiety bonded to the 3' end of A-U-G has been prepared with the aid of polynucleotide phosphorylase. This 3'-modified, chemically reactive A-U-G analog was used to probe the ribosomal codon binding site. The yield of the reaction depended strongly on the ribosomal source and was sensitive to salt-washing ribosomes. The major crosslinking product was identified to be protein S1. Since the reaction of this 3'-modified A-U-G programmed ribosomes for Met-tRNA-Met-M binding, it is concluded that protein S1 is located at or near the 3'-side of the ribosomal codon binding site. Images PMID:823527

  9. Hereditary sideroblastic anaemia due to a mutation in exon 10 of the erythroid 5-aminolaevulinate synthase gene.

    PubMed

    Edgar, A J; Wickramasinghe, S N

    1998-02-01

    DNA sequencing of the coding region of the erythroid 5-aminolaevulinate synthase (ALAS2) cDNA from a male with pyridoxine-responsive sideroblastic anaemia revealed a missense mutation C1622G and a closely linked polymorphism C1612A in exon 10 of the gene. Sequence analysis of the genomic DNA from other family members revealed that the proband's mother and daughter were heterozygous carriers of the mutation, consistent with the X-linked inheritance. The C1622G mutation results in a histidine to aspartic acid substitution at amino acid residue 524. The histidine residue is conserved in both the erythroid and housekeeping ALAS proteins in vertebrates, all other known ALAS proteins and other oxamine synthases that have pyridoxal 5'-phosphate as a co-factor. This histidine is located in a predicted loop, preceding a long alpha-helix region near the carboxy-terminus.

  10. Use of the pyrG gene as a food-grade selection marker in Monascus.

    PubMed

    Wang, Bo-hua; Xu, Yang; Li, Yan-ping

    2010-11-01

    Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5'-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.

  11. Morning sickness and vitamin B6 status of pregnant women.

    PubMed

    Schuster, K; Bailey, L B; Dimperio, D; Mahan, C S

    1985-01-01

    The relationship between the vitamin B6 status of 180 pregnant women and the incidence and degree of morning sickness experienced during the first trimester was investigated. There were no significant differences in plasma pyridoxal 5'-phosphate (PLP), erythrocyte aspartate aminotransferase (AspAT) activity, and stimulation of erythrocyte AspAT activity by exogenous PLP between subjects who experienced morning sickness and those who did not. No relationship was found between these indicators of vitamin B6 status and the degree of morning sickness experienced by this group during early pregnancy. There were no differences in the number of women who experienced morning sickness or in the number with different degrees of sickness when plasma levels of PLP, erythrocyte AspAT activity or stimulation by PLP were divided into upper and lower 50th percentile groups and compared. Therefore these data show no relationship between vitamin B6 status and the incidence or degree of morning sickness.

  12. Inhibition of Mycobacterium tuberculosis transaminase BioA by aryl hydrazines and hydrazides.

    PubMed

    Dai, Ran; Wilson, Daniel J; Geders, Todd W; Aldrich, Courtney C; Finzel, Barry C

    2014-03-03

    7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). Using biophysical fragment screening and structural characterization of compounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5'-phosphate (PLP) cofactor, forming a stable quinonoid. Analogous hydrazides also form covalent adducts that can be observed crystallographically but are incapable of inactivating the enzyme. In the X-ray crystal structures, small molecules induce unexpected conformational remodeling in the substrate binding site. We compared these conformational changes to those induced upon binding of the substrate (7-keto-8-aminopelargonic acid), and characterized the inhibition kinetics and the X-ray crystal structures of BioA with the hydrazine compound and analogues to unveil the mechanism of this reversible covalent modification.

  13. 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

  14. 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.

  15. 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.

  16. 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

    2015-11-11

    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.

  17. Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

    PubMed

    Revtovich, S V; Morozova, E A; Khurs, E N; Zakomirdina, L N; Nikulin, A D; Demidkina, T V; Khomutov, R M

    2011-05-01

    Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and β- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

  18. 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.

  19. 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

  20. Effects of exogenous glucose on carotenoid accumulation in tomato leaves.

    PubMed

    Mortain-Bertrand, Anne; Stammitti, Linda; Telef, Nadège; Colardelle, Patrice; Brouquisse, Renaud; Rolin, Dominique; Gallusci, Philippe

    2008-10-01

    To investigate the effect of carbohydrate on carotenoid accumulation in leaves, excised plants of tomato (Lycopersicum esculentum var. cerasiformae, wva 106) were supplied with glucose through the transpiration stream for 48 h. We report here that sugar accumulation in leaves led to a decrease of carotenoid content, which was related to the reduction of Chl. The decrease in carotenoid amount correlated with a sugar-induced repression of genes encoding enzymes of the carotenoid and of the Rohmer pathways. The lower 1-deoxy-D-xylulose-5-phosphate synthase transcript level probably leads to a decreased metabolic flux through the methylerythritol pathway and subsequently to a lower amount of substrate available for plastidic isoprenoid synthesis. Differences between responses of young (sink) and mature (source) leaves to carbohydrate accumulation are discussed.

  1. 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

  2. Chemical characterization of a prominent phosphomonoester resonance from mammalian brain. 31P and 1H NMR analysis at 4.7 and 14.1 tesla

    NASA Astrophysics Data System (ADS)

    Pettegrew, J. W.; Kopp, S. J.; Dadok, J.; Minshew, N. J.; Feliksik, J. M.; Glonek, T.; Cohen, M. M.

    A prominent 31P NMR resonance at 3.84 ppm in mammalian brain has been identified as ethanolamine phosphate. The identification was based on 1H and 31P NMR findings (including pH titrations) at 4.7 and 14.1 T, as well as thin-layer chromatography studies. We previously incorrectly assigned the 3.84 ppm resonance to ribose-5-phosphate. The incorrect assignment occurred because the two compounds have very similar 31P chemical shifts, and because we did not carefully consider the effects of counter ions and ionic strengths when interpreting the 31P chemical shifts. In separate preliminary studies we have demonstrated ethanolamine phosphate to be high in immature developing brain and in the degenerating brain of Alzheimer's and Huntington's disease patients. Ethanolamine phosphate may therefore serve as a sensitive marker of membrane phospholipid turnover for both in vitro and in vivo31P NMR studies.

  3. A simplified protein precipitation and filtration procedure for determining serum vitamin B6 by high-performance liquid chromatography.

    PubMed

    Rybak, Michael E; Pfeiffer, Christine M

    2009-05-01

    Protein precipitation followed by centrifuge filtration was tested as a simplified sample preparation procedure for quantifying pyridoxal 5'-phosphate (PLP) and 4-pyridoxic acid (4PA) in serum by high-performance liquid chromatography. Serum samples (n=160) were prepared by both centrifuge filtration and an established technique using traditional supernatant extraction with manual filtration. Bland-Altman bias analysis (95% confidence levels [CLs]) of the results showed a -1.3 (-2.2, -0.5)% difference in PLP values and a -6.2 (-7.3, -5.2)% difference in 4PA values using the simplified sample preparation. These deviations were found to be well within allowable biases on the basis of biologic variation.

  4. Characterization of superparamagnetic Fe3O4 nanoparticles by fluxgate magnetorelaxometry for use in biomedical applications

    NASA Astrophysics Data System (ADS)

    Ludwig, F.; Heim, E.; Schilling, M.; Enpuku, K.

    2008-04-01

    We have investigated the magnetorelaxometry (MRX) of various Fe3O4 nanoparticle suspensions utilizing a differential fluxgate setup. To estimate the distribution of the hydrodynamic size of the magnetic nanoparticles (MNPs) with organic shell, the cluster moment superposition model is applied. On a sample series of MNPs from the same batch diluted in water-glycerine mixtures of varying viscosity, it is demonstrated that the model is suited for the estimation of the distribution of the hydrodynamic size of MNPs and clusters. Since a MRX measurement lasts only a few seconds, it is also highly suited to study the aggregation kinetics. This is demonstrated on MNPs with silica shell suspended in 5% phosphate-buffered saline solution.

  5. The hydrophobic substituent in aminophospholipids affects the formation kinetics of their Schiff bases.

    PubMed

    Caldés, Catalina; Vilanova, Bartolomé; Adrover, Miquel; Donoso, Josefa; Muñoz, Francisco

    2013-04-01

    Schiff bases (SBs) are the initial products of non-enzymatic glycation reactions, which are associated to some diabetes-related diseases. In this work, we used physiological pH and temperature conditions to study the formation kinetics of the SBs of 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DPHE) and 1,2-dihexanoyl-sn-glycero-3-phospho-l-serine (DHPS) with various glycating compounds and with pyridoxal 5'-phosphate (an effective glycation inhibitor). Based on the obtained results, the hydrophobic environment simultaneously decreases the nucleophilic character of the amino group (k1) and increases its pKa, thereby increasing the formation rate of SB (kobs). Therefore, the presence of hydrophobic chains in aminophospholipids facilitates the formation and stabilization of SBs, and also, in a biological environment, their glycation. Additionally, the results confirm the inhibitory action of B6 vitamers on aminophospholipid glycation.

  6. Biochemical characterization and helix stabilizing properties of HSNP-C' from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    PubMed

    Celestina, F; Suryanarayana, T

    2000-01-19

    Helix stabilizing nucleoid protein HSNP-C' from the thermophilic archaeon Sulfolobus acidocaldarius has been characterized with respect to its interactions with nucleic acids by gel retardation assay, affinities to immobilized matrices, electron microscopy, and fluorescence titration. The amino acids implicated in the DNA binding site of the protein have been shown by selectively modifying specific amino acyl functional groups and looking at their effects on the DNA binding properties of the protein. Lysine, arginine, tryptophan, and tyrosine residues of the protein HSNP-C' were modified with pyridoxal-5-phosphate; 2,3-butanedione; BNPS-skatole; and tetranitromethane, respectively. The modification of residues was assessed according to standard procedures. The effect of the chemical modification on the function of the protein HSNP-C' with respect to DNA protein interactions was studied and the results indicate the definite involvement of tyrosines and also the significant involvement of the flanking tryptophan residues in the DNA binding domain on the protein.

  7. 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

  8. Syntheses of L-tyrosine-related amino acids by tyrosine phenol-lyase of Citrobacter intermedius.

    PubMed

    Nagasawa, T; Utagawa, T; Goto, J; Kim, C J; Tani, Y; Kumagai, H; Yamada, H

    1981-06-01

    Degradation of tyrosine to phenol, pyruvate and ammonia by tyrosine phenol-lyase from Citrobacter intermedius (formerly named Escherichia intermedia) is readily reversible at high concentrations of pyruvate and ammonia. Spectrophotometric studies indicate that ammonia is the first substrate which interacts with bound pyridoxal 5'-phosphate. Kinetic results show that pyruvate is the second substrate bound, hence phenol must be the third. When an appropriate phenol derivative is substituted for phenol, the corresponding tyrosine analogue can be synthesized. 3-Fluoro-, 2-fluoro-, 3-chloro-, 2-chloro-, 3-bromo-, 2-bromo-, 2-iodo-, 3-methyl-, 2-methyl- and 2-methoxy-L-tyrosines have been synthesized by this reaction. By using various phenol derivatives or tyrosine analogues as substrates, the substrate specificity of tyrosine phenol-lyase is investigated and the situation of its active site is discussed.

  9. Identification and characterization of a methionine γ-lyase in the calicheamicin biosynthetic cluster of Micromonospora echinospora.

    PubMed

    Song, Haigang; Xu, Ri; Guo, Zhihong

    2015-01-02

    CalE6 is a previously uncharacterized protein involved in the biosynthesis of calicheamicins in Micromonospora echinospora. It is a pyridoxal-5'-phosphate-dependent enzyme and exhibits high sequence homology to cystathionine γ-lyases and cystathionine γ-synthases. However, it was found to be active towards methionine and to convert this amino acid into α-ketobutyrate, ammonium, and methanethiol. The crystal structure of the cofactor-bound holoenzyme was resolved at 2.0 Å; it contains two active site residues, Gly105 and Val322, specific for methionine γ-lyases. Modeling of methionine into the active site allows identification of the active site residues responsible for substrate recognition and catalysis. These findings support that CalE6 is a putative methionine γ-lyase producing methanethiol as a building block in biosynthesis of calicheamicins.

  10. Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.

    PubMed Central

    Lund, V; Schmid, R; Rickwood, D; Hornes, E

    1988-01-01

    Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization. PMID:3205723

  11. Photocleavage of DNA and photofootprinting of E. coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines.

    PubMed Central

    Jeppesen, C; Buchardt, O; Henriksen, U; Nielsen, P E

    1988-01-01

    The long-wavelength ultraviolet (lambda approximately 420 nm) radiation induced reaction between 6-azido-2-methoxy-9-acridinylamines and supercoiled plasmid DNA results in single strand scissions and formation of covalent adducts (ratio approximately 1:10). By treating azidoacridine-photomodified DNA with piperidine at 90 degrees C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T greater than or equal to G greater than C much greater than A. The resulting DNA fragments migrate as 5'-phosphates in polyacrylamide gels. Photofootprinting of the binding site of RNA-polymerase on promoter DNA is demonstrated with an azido-9-acridinylamino-octamethylene-9-aminoacridine. Similar experiments using 9-amino-6-azido-2-methoxyacridine indicate that this reagent recognizes changes in the DNA conformation induced by RNA polymerase binding, in relation to open complex formation. Images PMID:3041368

  12. The structure of alanine racemase from Acinetobacter baumannii.

    PubMed

    Davis, Emily; Scaletti-Hutchinson, Emma; Opel-Reading, Helen; Nakatani, Yoshio; Krause, Kurt L

    2014-09-01

    Acinetobacter baumannii is an opportunistic Gram-negative bacterium which is a common cause of hospital-acquired infections. Numerous antibiotic-resistant strains exist, emphasizing the need for the development of new antimicrobials. Alanine racemase (Alr) is a pyridoxal 5'-phosphate dependent enzyme that is responsible for racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall, its inhibition is lethal to prokaryotes, making it an excellent antibiotic drug target. The crystal structure of A. baumannii alanine racemase (AlrAba) from the highly antibiotic-resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of AlrAba with alanine racemases from closely related bacteria demonstrates a conserved overall fold. The substrate entryway and active site of the enzymes were shown to be highly conserved. The structure of AlrAba will provide the template required for future structure-based drug-design studies.

  13. 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.

  14. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase

    PubMed Central

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  15. DNA ligases.

    PubMed

    Tabor, S

    2001-05-01

    DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.

  16. Coupled 5' nucleotide recognition and processivity in Xrn1-mediated mRNA decay.

    PubMed

    Jinek, Martin; Coyle, Scott M; Doudna, Jennifer A

    2011-03-04

    Messenger RNA decay plays a central role in the regulation and surveillance of eukaryotic gene expression. The conserved multidomain exoribonuclease Xrn1 targets cytoplasmic RNA substrates marked by a 5' monophosphate for processive 5'-to-3' degradation by an unknown mechanism. Here, we report the crystal structure of an Xrn1-substrate complex. The single-stranded substrate is held in place by stacking of the 5'-terminal trinucleotide between aromatic side chains while a highly basic pocket specifically recognizes the 5' phosphate. Mutations of residues involved in binding the 5'-terminal nucleotide impair Xrn1 processivity. The substrate recognition mechanism allows Xrn1 to couple processive hydrolysis to duplex melting in RNA substrates with sufficiently long single-stranded 5' overhangs. The Xrn1-substrate complex structure thus rationalizes the exclusive specificity of Xrn1 for 5'-monophosphorylated substrates, ensuring fidelity of mRNA turnover, and posits a model for translocation-coupled unwinding of structured RNA substrates.

  17. Untangling the role of one-carbon metabolism in colorectal cancer risk: a comprehensive Bayesian network analysis

    PubMed Central

    Myte, Robin; Gylling, Björn; Häggström, Jenny; Schneede, Jörn; Magne Ueland, Per; Hallmans, Göran; Johansson, Ingegerd; Palmqvist, Richard; Van Guelpen, Bethany

    2017-01-01

    The role of one-carbon metabolism (1CM), particularly folate, in colorectal cancer (CRC) development has been extensively studied, but with inconclusive results. Given the complexity of 1CM, the conventional approach, investigating components individually, may be insufficient. We used a machine learning-based Bayesian network approach to study, simultaneously, 14 circulating one-carbon metabolites, 17 related single nucleotide polymorphisms (SNPs), and several environmental factors in relation to CRC risk in 613 cases and 1190 controls from the prospective Northern Sweden Health and Disease Study. The estimated networks corresponded largely to known biochemical relationships. Plasma concentrations of folate (direct), vitamin B6 (pyridoxal 5-phosphate) (inverse), and vitamin B2 (riboflavin) (inverse) had the strongest independent associations with CRC risk. Our study demonstrates the importance of incorporating B-vitamins in future studies of 1CM and CRC development, and the usefulness of Bayesian network learning for investigating complex biological systems in relation to disease. PMID:28233834

  18. 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.

  19. 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.

  20. Crystal structures of [lamda] exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity

    SciTech Connect

    Zhang, Jinjin; McCabe, Kimberly A.; Bell, Charles E.

    2011-07-19

    The {lambda} exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5'-ended strand to form 5' mononucleotides and a long 3' overhang. The crystal structure of {lambda} exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of {lambda} exonuclease in complex with DNA at 1.88-{angstrom} resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5'-ended strand insert into the active site of one subunit of the trimer, while the 3'-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5' phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg{sup 2+} ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5' phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of {lambda} exonuclease operates not only at the level of the trimer, but also at the level of the monomer.

  1. 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.

  2. HDHD1, which is often deleted in X-linked ichthyosis, encodes a pseudouridine-5'-phosphatase.

    PubMed

    Preumont, Alice; Rzem, Rim; Vertommen, Didier; Van Schaftingen, Emile

    2010-10-15

    Pseudouridine, the fifth-most abundant nucleoside in RNA, is not metabolized in mammals, but is excreted intact in urine. The purpose of the present work was to search for an enzyme that would dephosphorylate pseudouridine 5'-phosphate, a potential intermediate in RNA degradation. We show that human erythrocytes contain a pseudouridine-5'-phosphatase displaying a Km ≤ 1 μM for its substrate. The activity of the partially purified enzyme was dependent on Mg2+, and was inhibited by Ca2+ and vanadate, suggesting that it belonged to the 'haloacid dehalogenase' family of phosphatases. Its low molecular mass (26 kDa) suggested that this phosphatase could correspond to the protein encoded by the HDHD1 (haloacid dehalogenase-like hydrolase domain-containing 1) gene, present next to the STS (steroid sulfatase) gene on human chromosome Xp22. Purified human recombinant HDHD1 dephosphorylated pseudouridine 5'-phosphate with a kcat of 1.6 s-1, a Km of 0.3 μM and a catalytic efficiency at least 1000-fold higher than that on which it acted on other phosphate esters, including 5'-UMP. The molecular identity of pseudouridine-5'-phosphatase was confirmed by the finding that its activity was negligible (<10% of controls) in extracts of B-cell lymphoblasts or erythrocytes from X-linked ichthyosis patients harbouring a combined deletion of the STS gene (the X-linked ichthyosis gene) and the HDHD1 gene. Furthermore, pseudouridine-5'-phosphatase activity was 1.5-fold higher in erythrocytes from women compared with men, in agreement with the HDHD1 gene undergoing only partial inactivation in females. In conclusion, HDHD1 is a phosphatase specifically involved in dephosphorylation of a modified nucleotide present in RNA.

  3. Expression of E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae.

    PubMed

    Sedlak; Ho

    2001-01-02

    The Escherichia coli araBAD operon consists of three genes encoding three enzymes that convert L-arabinose to D-xylulose-5 phosphate. In this paper we report that the genes of the E. coli araBAD operon have been expressed in Saccharomyces cerevisiae using strong promoters from genes encoding S. cerevisiae glycolytic enzymes (pyruvate kinase, phosphoglucose isomerase, and phosphoglycerol kinase). The expression of these cloned genes in yeast was demonstrated by the presence of the active enzymes encoded by these cloned genes and by the presence of the corresponding mRNAs in the new host. The level of expression of L-ribulokinase (araB) and L-ribulose-5-phosphate 4-epimerase (araD) in S. cerevisiae was relatively high, with greater than 70% of the activity of the enzymes in wild type E. coli. On the other hand, the expression of L-arabinose isomerase (araA) reached only 10% of the activity of the same enzyme in wild type E. coli. Nevertheless, S. cerevisiae, bearing the cloned L-arabinose isomerase gene, converted L-arabinose to detectable levels of L-ribulose during fermentation. However, S. cerevisiae bearing all three genes (araA, araB, and araD) was not able to produce detectable amount of ethanol from L-arabinose. We speculate that factors such as pH, temperature, and competitive inhibition could reduce the activity of these enzymes to a lower level during fermentation compared to their activity measured in vitro. Thus, the ethanol produced from L-arabinose by recombinant yeast containing the expressed BAD genes is most likely totally consumed by the cell to maintain viability.

  4. Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.

    PubMed

    Kass, Itamar; Hoke, David E; Costa, Mauricio G S; Reboul, Cyril F; Porebski, Benjamin T; Cowieson, Nathan P; Leh, Hervé; Pennacchietti, Eugenia; McCoey, Julia; Kleifeld, Oded; Borri Voltattorni, Carla; Langley, David; Roome, Brendan; Mackay, Ian R; Christ, Daniel; Perahia, David; Buckle, Malcolm; Paiardini, Alessandro; De Biase, Daniela; Buckle, Ashley M

    2014-06-24

    The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

  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. D-amino acids in the brain: the biochemistry of brain serine racemase.

    PubMed

    Baumgart, Florian; Rodríguez-Crespo, Ignacio

    2008-07-01

    It has been recently established that in various brain regions D-serine, the product of serine racemase, occupies the so-called 'glycine site' within N-methyl D-aspartate receptors. Mammalian brain serine racemase is a pyridoxal-5' phosphate-containing enzyme that catalyzes the racemization of L-serine to D-serine. It has also been shown to catalyze the alpha,beta-elimination of water from L-serine or D-serine to form pyruvate and ammonia. Serine racemase is included within the group of type II-fold pyridoxal-5' phosphate enzymes, together with many other racemases and dehydratases. Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli. It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP. In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such as glutathione. Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein interacting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability. In addition, it has also been proposed that serine racemase is regulated by phosphorylation. Thus, d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance. In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above.

  7. Hydrogen isotopic differences between C3 and C4 land plant lipids: consequences of compartmentation in C4 photosynthetic chemistry and C3 photorespiration.

    PubMed

    Zhou, Youping; Grice, Kliti; Stuart-Williams, Hilary; Hocart, Charles H; Gessler, Arthur; Farquhar, Graham D

    2016-12-01

    The (2) H/(1) H ratio of carbon-bound H in biolipids holds potential for probing plant lipid biosynthesis and metabolism. The biochemical mechanism underlying the isotopic differences between lipids from C3 and C4 plants is still poorly understood. GC-pyrolysis-IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry) measurement of the (2) H/(1) H ratio of leaf lipids from controlled and field grown plants indicates that the biochemical isotopic fractionation (ε(2) Hlipid_biochem ) differed between C3 and C4 plants in a pathway-dependent manner: ε(2) HC4  > ε(2) HC3 for the acetogenic pathway, ε(2) HC4  < ε(2) HC3 for the mevalonic acid pathway and the 1-deoxy-D-xylulose 5-phosphate pathway across all species examined. It is proposed that compartmentation of photosynthetic CO2 fixation into C4 mesophyll (M) and bundle sheath (BS) cells and suppression of photorespiration in C4 M and BS cells both result in C4 M chloroplastic pyruvate - the precursor for acetogenic pathway - being more depleted in (2) H relative to pyruvate in C3 cells. In addition, compartmentation in C4 plants also results in (i) the transferable H of NADPH being enriched in (2) H in C4 M chloroplasts compared with that in C3 chloroplasts for the 1-deoxy-D-xylulose 5-phosphate pathway pathway and (ii) pyruvate relatively (2) H-enriched being used for the mevalonic acid pathway in the cytosol of BS cells in comparison with that in C3 cells.

  8. 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.

  9. 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; ...

    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

  10. THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE PYRENOID PROTEIN OF EREMOSPHAERA VIRIDIS

    PubMed Central

    Holdsworth, Robert H.

    1971-01-01

    The pyrenoids of Eremosphaera viridis, a green alga, were isolated by density gradient centrifugation and their physical and enzymatic properties were studied. The ultraviolet absorption spectrum of sodium dodecyl sulfate (SDS) extracts of pyrenoids showed a single peak at a wavelength of 277 nm, indicating the presence of protein and the probable absence of nucleic acid. Upon electrophoresis on polyacrylamide gels containing SDS, 16 bands were resolved of which two, together, accounted for 90% of the total protein on the gels. The molecular weights of these two proteins were estimated to be 59,000 and 12,300 and the ratio by weight of the larger to the smaller protein was found to be 2:1. The physical and enzymatic properties of these two proteins were found to closely resemble the properties reported in the literature for the subunits of fraction I protein. Both pyrenoids and fraction I protein are localized in the chloroplast, and both have two principal protein components. The molecular weights and relative ratio of the two pyrenoid components are very similar to those of the two components of fraction I protein. The pyrenoid was found to contain a high specific activity of ribulose-1,5-diphosphate carboxylase which is the same enzymatic activity exhibited by fraction I protein. The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase activities was also noted in pyrenoid preparations. It is suggested that the pyrenoid contains fraction I protein and possibly other enzymes of the Calvin-Bassham carbon dioxide fixing pathway. PMID:5112653

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

    PubMed Central

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

    2016-01-01

    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. PMID:27295432

  12. His103 in yeast transketolase is required for substrate recognition and catalysis.

    PubMed

    Wikner, C; Meshalkina, L; Nilsson, U; Bäckström, S; Lindqvist, Y; Schneider, G

    1995-11-01

    Crystallographic studies of thiamin-diphosphate-dependent transketolase from Saccharomyces cerevisiae suggested the invariant active-site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis, H103 was replaced by alanine, asparagine and phenylalanine using site-directed mutagenesis. The crystallographic analysis of the mutant transketolases verified that no structural changes occurred as a consequence of the side-chain replacements. The residual catalytic activities of the mutant enzymes were 4.3% for the H103A, 2.4% for the H103N and 0.1% for the H103F mutant transketolase. Further kinetic analysis of the H103A and H103N mutant enzymes showed that the Km values for the coenzyme were increased by about eightfold. The Km values for the acceptor substrate ribose 5-phosphate were similar to the Km value for wild-type transketolase. However, the Km value for the donor substrate, xylulose 5-phosphate is increased more than tenfold in these two mutants. Circular dichroism spectra of the mutant enzymes also indicated a weaker binding of the donor substrate and/or a less stable reaction intermediate. These observations provide further evidence in support of the proposed role for this invariant residue in recognition of the donor substrate by forming a hydrogen bond between the side chain of H103 and the C1 hydroxyl group of the sugar phosphate. The significant decrease in catalytic activity suggests that this residue also facilitates catalysis, possibly by maintaining the optimal orientation of the donor substrate and reaction intermediates.

  13. Structure-Based Function Discovery of an Enzyme for the Hydrolysis of Phosphorylated Sugar Lactones

    PubMed Central

    Xiang, Dao Feng; Kolb, Peter; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Narindoshivili, Tamari; Williams, Howard J.; Shoichet, Brian K.; Almo, Steven C.; Raushel, Frank M.

    2012-01-01

    Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å away from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275 and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high energy intermediate (HEI) form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, d-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The kcat/Km values for the cobalt-substituted enzymes with these substrates are ~105 M−1 s−1. PMID:22313111

  14. A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia.

    PubMed

    Lane, Alexander; Boecklemann, Astrid; Woronuk, Grant N; Sarker, Lukman; Mahmoud, Soheil S

    2010-03-01

    We are developing Lavandula angustifolia (lavender) as a model system for investigating molecular regulation of essential oil (a mixture of mono- and sesquiterpenes) production in plants. As an initial step toward building the necessary 'genomics toolbox' for this species, we constructed two cDNA libraries from lavender leaves and flowers, and obtained sequence information for 14,213 high-quality expressed sequence tags (ESTs). Based on homology to sequences present in GenBank, our EST collection contains orthologs for genes involved in the 1-deoxy-D: -xylulose-5-phosphate (DXP) and the mevalonic acid (MVA) pathways of terpenoid biosynthesis, and for known terpene synthases and prenyl transferases. To gain insight into the regulation of terpene metabolism in lavender flowers, we evaluated the transcriptional activity of the genes encoding for 1-deoxy-D: -xylulose-5-phosphate synthase (DXS) and HMG-CoA reductase (HMGR), which represent regulatory steps of the DXP and MVA pathways, respectively, in glandular trichomes (oil glands) by real-time PCR. While HMGR transcripts were barely detectable, DXS was heavily expressed in this tissue, indicating that essential oil constituents are predominantly produced through the DXP pathway in lavender glandular trichomes. As anticipated, the linalool synthase (LinS)-the gene responsible for the production of linalool, a major constituent of lavender essential oil-was also strongly expressed in glands. Surprisingly, the most abundant transcript in floral glandular trichomes corresponded to a sesquiterpene synthase (cadinene synthase, CadS), although sesquiterpenes are minor constituents of lavender essential oils. This result, coupled to the weak activity of the MVA pathway (the main route for sesquiterpene production) in trichomes, indicates that precursor supply may represent a bottleneck in the biosynthesis of sesquiterpenes in lavender flowers.

  15. Structural analysis of the substrate recognition mechanism in O-phosphoserine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1.

    PubMed

    Nakamura, Takashi; Kawai, Yoshito; Kunimoto, Kohei; Iwasaki, Yuka; Nishii, Kaoru; Kataoka, Misumi; Ishikawa, Kazuhiko

    2012-09-07

    L-Cysteine is synthesized from O-acetyl-L-serine (OAS) and sulfide by O-acetylserine sulfhydrylase (OASS; EC 2.5.1.47) in plants and bacteria. O-phosphoserine sulfhydrylase (OPSS; EC 2.5.1.65) is a novel enzyme from the hyperthermophilic aerobic archaeon Aeropyrum pernix K1 (2003). OPSS can use OAS or O-phospho-L-serine (OPS) to synthesize L-cysteine. To elucidate the mechanism of the substrate specificity of OPSS, we analyzed three-dimensional structures of the active site of the enzyme. The active-site lysine (K127) of OPSS forms an internal Schiff base with pyridoxal 5'-phosphate. Therefore, crystals of the complexes formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with either OPS or OAS were prepared and examined by X-ray diffraction analysis. In contrast to that observed for OASS, no significant difference was seen in the overall structure between the free and complexed forms of OPSS. The side chains of T152, S153, and Q224 interacted with the carboxylate of the substrates, as a previous study has suggested. The side chain of R297 has been proposed to recognize the phosphate group of OPS. Surprisingly, however, the position of R297 was significantly unchanged in the complex of the OPSS K127A mutant with the external Schiff base, allowing enough space for an interaction with OPS. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates such as OPS.

  16. Human pyridoxal phosphatase. Molecular cloning, functional expression, and tissue distribution.

    PubMed

    Jang, Young Min; Kim, Dae Won; Kang, Tae-Cheon; Won, Moo Ho; Baek, Nam-In; Moon, Byung Jo; Choi, Soo Young; Kwon, Oh-Shin

    2003-12-12

    Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 microM and 1.52 s(-1), respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

  17. 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.

  18. Linkage of subunit interactions, structural changes, and energetics of coenzyme binding in tryptophan synthase.

    PubMed

    Wiesinger, H; Hinz, H J

    1984-10-09

    The energetics of binding of the coenzyme pyridoxal 5'-phosphate (PLP) to both the apo beta 2 subunit and the apo alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been investigated as a function of pH and temperature by direct microcalorimetric methods. At 25 degrees C, pH 7.5, the binding process proceeds in the time range of minutes and shows a biphasic heat output which permits resolution of the overall reaction into different reaction steps. Binding studies on the coenzyme analogues pyridoxal (PAL), pyridoxine 5'-phosphate (PNP), and pyridoxine (POL) to the protein as well as a comparison of these results with data from studies on PLP binding to epsilon-aminocaproic acid have led to a deconvolution of the complex heat vs. time curves into fast endothermic contributions from electrostatic interaction and Schiff base formation and slow exothermic contributions from the interactions between PLP and the binding domain. The pH-independent, large negative change in heat capacity of about -9.1 kJ/(mol of beta 2 X K) when binding PLP to beta 2 is indicative of major structural changes resulting from complex formation. The much smaller value of delta Cp = -1.7 kJ/(mol of beta 2 X K) for binding of PLP to alpha 2 beta 2 clearly demonstrates the energetic linkage of protein-protein and protein-ligand interactions. Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs. On the basis of these measurements a complete set of site-specific thermodynamic parameters has been established.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Crystal structure of the S187F variant of human liver alanine: glyoxylate [corrected] aminotransferase associated with primary hyperoxaluria type I and its functional implications.

    PubMed

    Oppici, Elisa; Fodor, Krisztian; Paiardini, Alessandro; Williams, Chris; Voltattorni, Carla Borri; Wilmanns, Matthias; Cellini, Barbara

    2013-08-01

    The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5'-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5'-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results.

  20. 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.

  1. Influence of Mg2+ and pH on n.m.r. spectra and radioligand binding of inositol 1,4,5-trisphosphate.

    PubMed Central

    White, A M; Varney, M A; Watson, S P; Rigby, S; Liu, C S; Ward, J G; Reese, C B; Graham, H C; Williams, R J

    1991-01-01

    We and others have shown that the binding of Ins(1,4,5)P3 to its receptor is pH-sensitive and can be inhibited by Mg2+. In the present study we have used 1H- and 31P-n.m.r. spectroscopy to study whether these effects results from increased ionization of Ins(1,4,5)P3 and a direct interaction with Mg2+ respectively. Under near-physiological conditions of ionic strength (100 mM-KCl), three ionizable groups were observed. The pH titration curve of the 1-phosphate was monophasic, with a pKa of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pKa values for the 4-phosphate determined by 31P-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. 1H- and 31P-n.m.r. measurements suggest that Mg2+ binds weakly to Ins(1,4,5)P3 at physiological pH. Mg2+ non-competitively inhibited binding of Ins(1,4,5)P3 to its receptor in rat cerebellum and bovine adrenal cortex. Inhibition curves for rat cerebellum at pH 7.1 and 8.5, and also for bovine adrenal cortex at pH 8.5, appeared to be monophasic, with IC50 values (concn. of displacer giving 50% inhibition of specific binding) of 214 microM, 572 microM and 9.1 mM respectively. Scatchard analysis revealed that Mg2+ inhibited binding of Ins(1,4,5)P3 to bovine adrenal cortex at pH 8.5 in a non-competitive manner. Our results suggest that the previously reported pH-sensitivity of the binding of Ins(1,4,5)P3 may be caused by ionization of the phosphate groups in positions 4 and 5, and that the ability of Mg2+ to inhibit the binding of Ins(1,4,5)P3 is not mediated by direct chelation but through a site located on, or close to, the Ins(1,4,5)P3 receptor. Inhibition by Mg2+ is pH-sensitive and can vary at least 10-fold between tissues, suggesting possible receptor heterogeneity. Mg2+ may exert an important regulatory control on the release of Ca2+ by Ins(1,4,5)P3. PMID:1654891

  2. Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

    PubMed Central

    Hespell, R B; Odelson, D A

    1978-01-01

    During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent

  3. Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch fermentation

    PubMed Central

    2010-01-01

    Background Gamma-aminobutyric acid is a major inhibitory neurotransmitter in mammalian brains, and has several well-known physiological functions. Lactic acid bacteria possess special physiological activities and are generally regarded as safe. Therefore, using lactic acid bacteria as cell factories for gamma-aminobutyric acid production is a fascinating project and opens up a vast range of prospects for making use of GABA and LAB. We previously screened a high GABA-producer Lactobacillus brevis NCL912 and optimized its fermentation medium composition. The results indicated that the strain showed potential in large-scale fermentation for the production of gamma-aminobutyric acid. To increase the yielding of GABA, further study on the fermentation process is needed before the industrial application in the future. In this article we investigated the impacts of pyridoxal-5'-phosphate, pH, temperature and initial glutamate concentration on gamma-aminobutyric acid production by Lactobacillus brevis NCL912 in flask cultures. According to the data obtained in the above, a simple and effective fed-batch fermentation method was developed to highly efficiently convert glutamate to gamma-aminobutyric acid. Results Pyridoxal-5'-phosphate did not affect the cell growth and gamma-aminobutyric acid production of Lb. brevis NCL912. Temperature, pH and initial glutamate concentration had significant effects on the cell growth and gamma-aminobutyric acid production of Lb. brevis NCL912. The optimal temperature, pH and initial glutamate concentration were 30-35°C, 5.0 and 250-500 mM. In the following fed-batch fermentations, temperature, pH and initial glutamate concentration were fixed as 32°C, 5.0 and 400 mM. 280.70 g (1.5 mol) and 224.56 g (1.2 mol) glutamate were supplemented into the bioreactor at 12 h and 24 h, respectively. Under the selected fermentation conditions, gamma-aminobutyric acid was rapidly produced at the first 36 h and almost not produced after then. The gamma

  4. Structure And Mutagenic Conversion of E(1) Dehydrase: at the Crossroads of Dehydration, Amino Transfer, And Epimerization

    SciTech Connect

    Smith, P.; Szu, P.-H.; Bui, C.; Liu, H.-w.; Tsai, S.-C.

    2009-05-26

    Pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are highly versatile coenzymes whose importance is well recognized. The capability of PLP/PMP-dependent enzymes to catalyze a diverse array of chemical reactions is attributed to fine-tuning of the cofactor-substrate interactions in the active site. CDP-6-deoxy-l-threo-d-glycero-4-hexulose 3-dehydrase (E1), along with its reductase (E{sub 3}), catalyzes the C-3 deoxygenation of CDP-4-keto-6-deoxy-d-glucose to form the dehydrated product, CDP-4-keto-3,6-dideoxy-d-glucose, in the ascarylose biosynthetic pathway. This product is the progenitor to most 3,6-dideoxyhexoses, which are the major antigenic determinants of many Gram-negative pathogens. The dimeric [2Fe-2S] protein, E{sub 1}, cloned from Yersinia pseudotuberculosis, is the only known enzyme whose catalysis involves the direct participation of PMP in one-electron redox chemistry. E{sub 1} also contains an unusual [2Fe-2S] cluster with a previously unknown binding motif (C-X{sub 57}-C-X{sub 1}-C-X{sub 7}-C). Herein we report the first X-ray crystal structure of E{sub 1}, which exhibits an aspartate aminotransferase (AAT) fold. A comparison of the E{sub 1} active site architecture with homologous structures uncovers residues critical for the dehydration versus transamination activity. Site-directed mutagenesis of four E{sub 1} residues, D194H, Y217H, H220K, and F345H, converted E{sub 1} from a PMP-dependent dehydrase to a PLP/glutamate-dependent aminotransferase. The E{sub 1} quadruple mutant, having been conferred this altered enzyme activity, can transaminate the natural substrate to CDP-4,6-dideoxy-4-amino-d-galactose without E{sub 3}. Taken together, these results provide the molecular basis of the functional switch of E{sub 1} toward dehydration, epimerization, and transamination. The insights gained from these studies can be used for the development of inhibitors of disease-relevant PLP/PMP-dependent enzymes.

  5. Effects of forskolin on electrical behaviour of myenteric neurones in guinea-pig small intestine.

    PubMed Central

    Nemeth, P R; Palmer, J M; Wood, J D; Zafirov, D H

    1986-01-01

    The actions of forskolin on electrical behaviour of myenteric neurones were investigated with intracellular recording methods in guinea-pig small intestine. The actions of forskolin were: membrane depolarization, increased input resistance, suppression of post-spike hyperpolarizing potentials and repetitive spike discharge. These effects occurred always in AH/Type 2 myenteric neurones and never in the cells classified as S/Type 1. Reversal potentials for the depolarizing effects were near the estimated potassium equilibrium potential. Analyses based on the 'constant field equation' indicated that the permeability ratios of K+ to other permeant ionic species were reduced by forskolin. Pretreatment of the neurones with a phosphodiesterase inhibitor potentiated the effects of forskolin. The results suggest that activation of adenylate cyclase by forskolin and subsequent elevation of intraneuronal adenosine 3',5'-phosphate (cyclic AMP) mimic slow synaptic excitation in AH/Type 2 myenteric neurones. They support the possibility that cyclic AMP functions as a second messenger in signal transduction which appears to involve closure of calcium-dependent K+ channels and other membrane changes that lead to depolarization and a dramatic increase in the excitability of the neurones. PMID:2432235

  6. On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase.

    PubMed

    Giovannercole, F; Mérigoux, C; Zamparelli, C; Verzili, D; Grassini, G; Buckle, M; Vachette, P; De Biase, D

    2017-01-05

    Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.

  7. Fabrication of luminescent hydroxyapatite nanorods through surface-initiated RAFT polymerization: Characterization, biological imaging and drug delivery applications

    NASA Astrophysics Data System (ADS)

    Heng, Chunning; Zheng, Xiaoyan; Liu, Meiying; Xu, Dazhuang; Huang, Hongye; Deng, Fengjie; Hui, Junfeng; Zhang, Xiaoyong; Wei, Yen

    2016-11-01

    Hydroxyapatite nanomaterials as an important class of nanomaterials, have been widely applied for different biomedical applications for their excellent biocompatibility, biodegradation potential and low cost. In this work, hydroxyapatite nanorods with uniform size and morphology were prepared through hydrothermal synthesis. The surfaces of these hydroxyapatite nanorods are covered with hydrophobic oleic acid, making them poor dispersibility in aqueous solution and difficult for biomedical applications. To overcome this issue, a simple surface initiated polymerization strategy has been developed via combination of the surface ligand exchange and reversible addition fragmentation chain transfer (RAFT) polymerization. Hydroxyapatite nanorods were first modified with Riboflavin-5-phosphate sodium (RPSSD) via ligand exchange reaction between the phosphate group of RPSSD and oleic acid. Then hydroxyl group of nHAp-RPSSD was used to immobilize chain transfer agent, which was used as the initiator for surface-initiated RAFT polymerization. The nHAp-RPSSD-poly(IA-PEGMA) nanocomposites were characterized by means of 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, fluorescence spectroscopy and thermal gravimetric analysis in detailed. The biocompatibility, biological imaging and drug delivery of nHAp-RPSSD-poly(IA-PEGMA) were also investigated. Results showed that nHAp-RPSSD-poly(IA-PEGMA) exhibited excellent water dispersibility, desirable optical properties, good biocompatibility and high drug loading capability, making them promising candidates for biological imaging and controlled drug delivery applications.

  8. Public health significance of elevated homocysteine.

    PubMed

    Selhub, Jacob

    2008-06-01

    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 mild elevations of homocysteine in plasma are a risk factor for occlusive vascular disease. In the Framingham studies, we have shown that plasma homocysteine concentration is inversely related to the intake and plasma levels of folate and vitamin B6 as well as vitamin B12 plasma levels. Almost two-thirds of the prevalence of high homocysteine is attributable to low vitamin status or intake. Elevated homocysteine concentrations in plasma are a risk factor for prevalence of extracranial carotid-artery stenosis > or = 25% in both men and women. Prospectively elevated plasma homocysteine is associated with increased total and cardiovascular mortality, increased incidence of stroke, increased incidence of dementia and Alzheimer's disease, increased incidence of bone fracture, and higher prevalence of chronic heart failure. It was also shown that elevated plasma homocysteine is a risk factor for preeclampsia and maybe neural tube defects (NTD). This multitude of relationships between elevated plasma homocysteine and diseases that afflict the elderly, pregnant women, and the embryo points to the existence ofa common denominator which may be responsible for these diseases. Whether this denominator is homocysteine itself or homocysteine is merely a marker, remains to be determined.

  9. Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

    PubMed Central

    Bartholeyns, J; Baudhuin, P

    1977-01-01

    Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor. PMID:19011

  10. Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

    SciTech Connect

    Boonsri, Pornthip; Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng; Herdendorf, Timothy J.; Miziorko, Henry M.; Hannongbua, Supa; Sem, Daniel S.

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Natural and synthetic inhibitors of human phosphomevalonate kinase identified. Black-Right-Pointing-Pointer Virtual screening yielded a hit rate of 15%, with inhibitor K{sub d}'s of 10-60 {mu}M. Black-Right-Pointing-Pointer NMR studies indicate significant protein conformational changes upon binding. -- Abstract: Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based {sup 1}H-{sup 15}N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K{sub d}). Tight binding compounds with K{sub d}'s ranging from 6-60 {mu}M were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.

  11. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    SciTech Connect

    Timofeev, V. I. Abramchik, Yu. A. Zhukhlistova, N. E. Kuranova, I. P.

    2015-09-15

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6{sub 3}22 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  12. Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

    PubMed

    Miroshnikova, A D; Kuznetsova, A A; Kuznetsov, N A; Fedorova, O S

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

  13. Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

    2013-07-01

    Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

  14. Health-hazard evaluation report HETA 86-456-1877, South Texas Nuclear Project, Wadsworth, Texas

    SciTech Connect

    Sinks, T.H.; Hartle, R.W.

    1988-03-01

    An evaluation was made of an outbreak of dermatitis among workers at the South Texas Nuclear Project construction site, Wadsworth, Texas. The dermatitis occurred ten times more frequently among carpenters than other laborers, with the incidence in 1986 being 250% greater than it was in 1985. Some workers demonstrated pruritic, macular/papular lesions. Carpenters working on the inside of the power-project buildings had a higher incidence of skin disease than those employed on the outside of the buildings. Samples of plywood and lumber treated with fire-retardant indicated that they contained 3 and 5% phosphate, respectively. Arsenic was not detected but formaldehyde was detected at 59 parts per million. General environmental air samples were taken with no evidence found of airborne phosphate, melamine, dicyandiamide, or formaldehyde. Concentrations of total particulates ranged from 0.1 to 0.6mg/m/sup 3/. The authors conclude that the workers were probably suffering from a contact dermatitis. The authors recommend specific precautions.

  15. Differential inhibition/inactivation of mitochondrial complex I implicates its alteration in malignant cells.

    PubMed

    Ghosh, A; Bera, S; Ghosal, S; Ray, S; Basu, A; Ray, M

    2011-09-01

    Methylglyoxal strongly inhibited mitochondrial respiration of a wide variety of malignant tissues including sarcoma of mice, whereas no such significant effect was noted on mitochondrial respiration of normal tissues with the exception of cardiac cells. This inhibition by methylglyoxal was found to be at the level of mitochondrial complex I (NADH dehydrogenase) of the electron transport chain. L-Lactaldehyde, which is structurally and metabolically related to methylglyoxal, could protect against this inhibition. NADH dehydrogenase of submitochondrial particles of malignant and cardiac cells was inhibited by methylglyoxal. This enzyme of these cells was also inactivated by methylglyoxal. The possible involvement of lysine residue(s) for the activity of NADH dehydrogenase was also investigated by using lysine-specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal 5' phosphate (PP). Inactivation of NADH dehydrogenase by both TNBS and PP convincingly demonstrated the involvement of lysine residue(s) for the activity of the sarcoma and cardiac enzymes, whereas both TNBS and PP failed to inactivate the enzymes of skeletal muscle and liver. Together these studies demonstrate a specific effect of methylglyoxal on mitochondrial complex I of malignant cells and importantly some distinct alteration of this complex in cancer cells.

  16. Metabolic flux analysis of Saccharomyces cerevisiae in a sealed winemaking fermentation system.

    PubMed

    Li, Hua; Su, Jing; Ma, Wen; Guo, Anque; Shan, Zuhua; Wang, Hua

    2015-03-01

    A sealed fermentation (SF) system and an anaerobic fermentation (AF) system (under normal atmospheric pressure conditions) were employed to study the influence of endogenous carbon dioxide (CO2) on the metabolism of Saccharomyces cerevisiae. The results showed that the fermentation stopped when 82.0 g L(-1) glucose was consumed and the endogenously produced CO2: pressure reached to 14.3 MPa in SF system, while the sugar was used up during AF. The total yeast viable count in the end of AF was higher than that of SF. It was also observed that the ethanol yield in AF and SF was similar, the glycerol yield in AF was 1.26 times higher than that in SF, while the succinic acid and acetic acid yields in SF were 24.7 and 26 times higher than that in AF, respectively. Additionally, this work provides a stoichiometric model used for metabolic flux analysis of S. cerevisiae to compare the flux distribution in SF and AF. The results showed that CO2 had an important effect on the pathways of oxaloacetic acid formation from pyruvic acid and ribose-5-phosphate formation from glucose-6-phosphate. However, the pathway of ethanol formation from pyruvic acid (decarboxylation reaction), catalyzed by pyruvate decarboxylase, was insensitive to CO2.

  17. Comparative metabolic flux analysis of an Ashbya gossypii wild type strain and a high riboflavin-producing mutant strain.

    PubMed

    Jeong, Bo-Young; Wittmann, Christoph; Kato, Tatsuya; Park, Enoch Y

    2015-01-01

    In the present study, we analyzed the central metabolic pathway of an Ashbya gossypii wild type strain and a riboflavin over-producing mutant strain developed in a previous study in order to characterize the riboflavin over-production pathway. (13)C-Metabolic flux analysis ((13)C-MFA) was carried out in both strains, and the resulting data were fit to a steady-state flux isotopomer model using OpenFLUX. Flux to pentose-5-phosphate (P5P) via the pentose phosphate pathway (PPP) was 9% higher in the mutant strain compared to the wild type strain. The flux from purine synthesis to riboflavin in the mutant strain was 1.6%, while that of the wild type strain was only 0.1%, a 16-fold difference. In addition, the flux from the cytoplasmic pyruvate pool to the extracellular metabolites, pyruvate, lactate, and alanine, was 2-fold higher in the mutant strain compared to the wild type strain. This result demonstrates that increased guanosine triphosphate (GTP) flux through the PPP and purine synthesis pathway (PSP) increased riboflavin production in the mutant strain. The present study provides the first insight into metabolic flux through the central carbon pathway in A. gossypii and sets the foundation for development of a quantitative and functional model of the A. gossypii metabolic network.

  18. [Sequence-specific interaction of pyrimidine oligonucleotides with double-stranded DNA at acidic pH complexes of different types].

    PubMed

    Brossalina, E B; Demchenko, E N; Demchenko, Iu N; Vlassov, V V

    2009-01-01

    The interaction of pyrimidine oligonucleotides (OLN(15) and OLN(6)) and their alkylating derivatives bearing 4-(3-amino)-N-methyl and N-2-chloroethyl (RCl) aniline residues at the 5'-phosphate with a fragment of the human gamma-interferon gene was studied. In the presence of 150 mM NaCl at pH 5.4, the yield of dsDNA alkylation was 60% for RCl-OLN(15) and 10% for RCl-OLN(6); at pH 4.0 in the presence of 150 mM NaCl and 10 mM MgCl2, the yield of the dsDNA modification product was 100% for RCl-OLN(6) and 50% for RCl-OLN(15). It was shown by native electrophoresis that OLN(15) could form with the target dsDNA complexes of two types in the presence of magnesium ions at pH 4.0. One of the complexes was stable at pH 5.4 in the presence of magnesium ions, whereas the other was not. We found that only the complex stable in 10 mM Mg(OAc)2, pH 5.4, was effectively alkylated.

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

    PubMed Central

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

    2016-01-01

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

  20. Decreased coenzyme A levels in ridA mutant strains of Salmonella enterica result from inactivated serine hydroxymethyltransferase.

    PubMed

    Flynn, Jeffrey M; Christopherson, Melissa R; Downs, Diana M

    2013-08-01

    The RidA/Yer057/UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have enamine deaminase activity, and in their absence the reactive 2-aminoacrylate (2-AA) accumulates and inactivates at least some pyridoxal 5'-phosphate (PLP)-containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2-AA was a ubiquitous cellular stressor that was generated in central metabolism. Phenotypically, strains of S. enterica that lack RidA accumulated significantly more pyruvate in the growth medium than wild-type strains. Here we dissected this ridA mutant phenotype and showed it was an indirect consequence of damage to serine hydroxymethyltransferase (GlyA; E.C. 2.1.2.1). The results here identified a fourth PLP enzyme as a target of enamine stress in Salmonella.

  1. Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation.

    PubMed

    Wu, Fang; Gehring, Heinz

    2009-02-01

    Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC(50) up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CH(X))(4)-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO.

  2. SEM/EDX and confocal microscopy analysis of novel and conventional enteric-coated systems.

    PubMed

    Liu, Fang; Lizio, Rosario; Schneider, Uwe J; Petereit, Hans-Ulrich; Blakey, Peter; Basit, Abdul W

    2009-03-18

    A novel double coating enteric system (comprising an inner layer of neutralised EUDRAGIT) L 30 D-55 and organic acid, and an outer layer of standard EUDRAGIT) L 30 D-55) was developed to provide fast dissolution in proximal small intestinal conditions. The mechanisms involved in the dissolution of the double coating were investigated and compared with a conventional single layer enteric coating and an hypromellose (HPMC) sub-coated enteric system. Rates of drug release from coated prednisolone pellets were established using USP II dissolution methods (0.1M HCl for 2h and subsequently pH 5.5 phosphate buffer) and the coating dissolution process was illustrated using confocal laser scanning microscopy (CLSM). The distribution of sodium, as a representative ion, in the double-coating system during dissolution was determined using scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX). The double-coating system showed faster dissolution compared to the single coating and the HPMC sub-coated system in pH 5.5 buffer. The dissolution process of the double-coating was unusual; the inner coat dissolved before the outer coat and this accelerated the dissolution of the outer coat. During dissolution, sodium ions diffused from the inner coat to the outer coat. This migration of ions and the increased ionic strength and buffer capacity of the inner coat contribute to the rapid dissolution of the double-coating system.

  3. Steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer and pyrene for alkaline phosphatase fluorescent sensing.

    PubMed

    Song, Chunxia; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Jianbo; Huang, Jin; Zhou, Maogui; Guo, Xiaochen

    2016-03-05

    We herein report a strategy for sensitive alkaline phosphatase (ALP) fluorescent sensing based on steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer (polyβ-CD) and pyrene. The fluorescence of pyrene was enhanced more than 10 times through supramolecular assembly with polyβ-CD. The 5'-phosphorylated dsDNA probe with pyrene attached on the 3'-terminal could be cleaved by λ exonuclease (λ exo), yielding pyrene attached on mononucleotides. Pyrene attached on mononucleotides could easily enter the cavity of polyβ-CD, resulting in fluorescence enhancement. When ALP was introduced, it could remove 5'-phosphate groups from dsDNA and then prevented the cleavage of dsDNA. Pyrene attached on dsDNA was difficult to enter the cavity of polyβ-CD because of steric hindrance, resulting in an inconspicuous fluorescence enhancement. Owing to the excellent fluorescence enhancement during steric hindrance regulated supramolecular assembly, excellent performance of the assay method was achieved for ALP with a detection limit of 0.04 Um L(-1). The detection limit was superior or comparable with the reported methods. Besides, this method was simple in design, avoiding double-labeling of probe.

  4. A new glycation product ‘norpronyl-lysine,’ and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen

    PubMed Central

    Bullock, Peter T. B.; Reid, David G.; Ying Chow, W.; Lau, Wendy P. W.; Duer, Melinda J.

    2014-01-01

    NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-13C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13C–13C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13C5-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products. PMID:27919030

  5. Antitumour, antimicrobial and catalytic activity of gold nanoparticles synthesized by different pH propolis extracts

    NASA Astrophysics Data System (ADS)

    Gatea, Florentina; Teodor, Eugenia Dumitra; Seciu, Ana-Maria; Covaci, Ovidiu Ilie; Mănoiu, Sorin; Lazăr, Veronica; Radu, Gabriel Lucian

    2015-07-01

    The Romanian propolis was extracted in five different media, respectively, in water (pH 6.8), glycine buffer (pH 2.5), acetate buffer (pH 5), phosphate buffer (pH 7.4) and carbonate buffer (pH 9.2). The extracts presented different amounts of flavonoids and phenolic acids, increasing pH leading to higher concentrations of active compounds. Five variants of gold nanoparticles suspensions based on different pH Romanian propolis aqueous extracts were successfully synthesized. The obtained nanoparticles presented dimensions between 20 and 60 nm in dispersion form and around 18 nm in dried form, and different morphologies (spherical, hexagonal, triangular). Fourier transform infrared spectroscopy proved the attachment of organic compounds from propolis extracts to the colloidal gold suspensions and X-ray diffraction certified that the suspensions contain metallic gold. The obtained propolis gold nanoparticles do not exhibit any antibacterial or antifungal activity, but presented different catalytic activities and toxicity on tumour cells.

  6. The 1.9 A Structure of the Branched-Chain Amino-Acid Transaminase (IlvE) from Mycobacterium tuberculosis

    SciTech Connect

    Tremblay, L.; Blanchard, J

    2009-01-01

    Unlike mammals, bacteria encode enzymes that synthesize branched-chain amino acids. The pyridoxal 5'-phosphate-dependent transaminase performs the final biosynthetic step in these pathways, converting keto acid precursors into {alpha}-amino acids. The branched-chain amino-acid transaminase from Mycobacterium tuberculosis (MtIlvE) has been crystallized and its structure has been solved at 1.9 {angstrom} resolution. The MtIlvE monomer is composed of two domains that interact to form the active site. The biologically active form of IlvE is a homodimer in which each monomer contributes a substrate-specificity loop to the partner molecule. Additional substrate selectivity may be imparted by a conserved N-terminal Phe30 residue, which has previously been observed to shield the active site in the type IV fold homodimer. The active site of MtIlvE contains density corresponding to bound PMP, which is likely to be a consequence of the presence of tryptone in the crystallization medium. Additionally, two cysteine residues are positioned at the dimer interface for disulfide-bond formation under oxidative conditions. It is unknown whether they are involved in any regulatory activities analogous to those of the human mitochondrial branched-chain amino-acid transaminase.

  7. Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

    PubMed

    Sherman, J A; Matson, S W

    1994-10-21

    Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

  8. Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1.

    PubMed

    Setayesh, Neda; Sabouri-Shahrbabak, Saleheh; Bakherad, Hamid; Sepehrizadeh, Zargham

    2013-12-01

    DNA ligases join 3' hydroxyl and 5' phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.

  9. ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

    PubMed

    Petrova, T; Bezsudnova, E Y; Boyko, K M; Mardanov, A V; Polyakov, K M; Volkov, V V; Kozin, M; Ravin, N V; Shabalin, I G; Skryabin, K G; Stekhanova, T N; Kovalchuk, M V; Popov, V O

    2012-12-01

    DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

  10. Detection of T4 polynucleotide kinase activity with immobilization of TiO2 nanotubes and amplification of Au nanoparticles.

    PubMed

    Wang, Guangfeng; He, Xiuping; Xu, Gang; Chen, Ling; Zhu, Yanhong; Zhang, Xiaojun; Wang, Lun

    2013-05-15

    Determination of nucleotide kinase activity is valuable due to its importance in regulating nucleic acid metabolism. Herein, we describe a strategy for simply and accurately determining nucleotide kinase activity by TiO2 nanotubes mediated signal transition and Au nanoparticles amplification. In this method, DNA containing 5'-hydroxyl group is self-assembled onto a gold electrode and used as a substrate for T4 polynucleotide kinase (PNK). By the specific immobilization affinity of TiO2 nanotubes with the phosphorylated DNA, TiO2 nanotubes were linked with phosphorylated substrate DNA on the electrode. And then Au nanoparticles modified 5'-phosphate DNA was conjugated with the TiO2 nanotubes and hybridized with methylene blue labeled signal DNA. Because gold nanoparticles have high loading of signal indicator methylene blue, the electrochemical signal is generated and amplified. It presents an excellent performance with wide linear range and low detection limit. Additionally, inhibition effects of some salts have also been investigated. The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.

  11. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  12. Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages

    PubMed Central

    Hollenbaugh, Joseph A.; Montero, Catherine; Schinazi, Raymond F.; Munger, Joshua; Kim, Baek

    2016-01-01

    We evaluated cellular metabolism profiles of HIV-1 and HIV-2 infected primary human monocyte-derived macrophages (MDMs). First, HIV-2 GL-AN displays faster production kinetics and greater amounts of virus as compared to HIV-1s: YU-2, 89.6 and JR-CSF. Second, quantitative LC–MS/MS metabolomics analysis demonstrates very similar metabolic profiles in glycolysis and TCA cycle metabolic intermediates between HIV-1 and HIV-2 infected macrophages, with a few notable exceptions. The most striking metabolic change in MDMs infected with HIV-2 relative to HIV-1-infected MDMs was the increased levels of quinolinate, a metabolite in the tryptophan catabolism pathway that has been linked to HIV/AIDS pathogenesis. Third, both HIV-1 and HIV-2 infected MDMs showed elevated levels of ribose-5-phosphate, a key metabolic component in nucleotide biosynthesis. Finally, HIV-2 infected MDMs display increased dNTP concentrations as predicted by Vpx-mediated SAMHD1 degradation. Collectively, these data show differential metabolic changes during HIV-1 and HIV-2 infection of macrophages. PMID:26895248

  13. Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

    PubMed

    Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

    2014-07-01

    Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos.

  14. Organic cofactors in the metabolism of Dehalococcoides mccartyi strains

    PubMed Central

    Schipp, Christian J.; Marco-Urrea, Ernest; Kublik, Anja; Seifert, Jana; Adrian, Lorenz

    2013-01-01

    Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes. PMID:23479751

  15. Solution activity product (KFAP) and simultaneous demineralization-remineralization in bovine tooth enamel and hydroxyapatite pellets

    SciTech Connect

    Fox, J.L.; Iyer, B.V.; Higuchi, W.I.; Hefferren, J.J.

    1983-11-01

    The effects of changing the ion activity product of the remineralization solution at pH 4.5 (pKFAP 108-118) on the remineralization behavior of demineralized bovine tooth enamel and hydroxyapatite pellets have been studied. Solutions containing calcium-4.5, phosphate, and fluoride in acetate buffers were used. The /sup 45/Ca/F molar ratios indicated the formation of fluoridated hydroxyapatite in the enamel or the pellet when the pKFAP values for remineralizing solutions were less than 112. When the pKFAP values were greater than 112, the /sup 45/Ca/F ratios were found to be much less than 5. Also, when the pKFAP values were large (greater than 112), the remineralization patterns based on the fluoride distribution in the tooth (or pellet) were found to be different than when the pKFAP values were small (less than 112). The hypothesis that a pKFAP value of 112 is the demarcation between remineralization only and simultaneous dissolution-remineralization has been proposed based on these results.

  16. The Impact of Soy Isoflavones on MCF-7 and MDA-MB-231 Breast Cancer Cells Using a Global Metabolomic Approach

    PubMed Central

    Uifălean, Alina; Schneider, Stefanie; Gierok, Philipp; Ionescu, Corina; Iuga, Cristina Adela; Lalk, Michael

    2016-01-01

    Despite substantial research, the understanding of the chemopreventive mechanisms of soy isoflavones remains challenging. Promising tools, such as metabolomics, can provide now a deeper insight into their biochemical mechanisms. The purpose of this study was to offer a comprehensive assessment of the metabolic alterations induced by genistein, daidzein and a soy seed extract on estrogen responsive (MCF-7) and estrogen non-responsive breast cancer cells (MDA-MB-231), using a global metabolomic approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that all test compounds induced a biphasic effect on MCF-7 cells and only a dose-dependent inhibitory effect on MDA-MB-231 cells. Proton nuclear magnetic resonance (1H-NMR) profiling of extracellular metabolites and gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites confirmed that all test compounds shared similar metabolic mechanisms. Exposing MCF-7 cells to stimulatory concentrations of isoflavones led to increased intracellular levels of 6-phosphogluconate and ribose 5-phosphate, suggesting a possible upregulation of the pentose phosphate pathway. After exposure to inhibitory doses of isoflavones, a significant decrease in glucose uptake was observed, especially for MCF-7 cells. In MDA-MB-231 cells, the glutamine uptake was significantly restricted, leading to alterations in protein biosynthesis. Understanding the metabolomic alterations of isoflavones represents a step forward in considering soy and soy derivates as functional foods in breast cancer chemoprevention. PMID:27589739

  17. Structural, biochemical, and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis.

    PubMed

    Covarrubias, Adrian Suarez; Högbom, Martin; Bergfors, Terese; Carroll, Paul; Mannerstedt, Karin; Oscarson, Stefan; Parish, Tanya; Jones, T Alwyn; Mowbray, Sherry L

    2008-09-05

    Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C(gamma) in the formation of the alphabeta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for grow