WorldWideScience

Sample records for 1-deoxy-d-xylulose 5-phosphate reductoisomerase

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

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    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.

  2. 1-deoxy-D-xylulose-5-phosphate reductoisomerases, and methods of use

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    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.

  3. 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin

    Dhiman, Rakesh K.; Schaeffer, Merrill L.; Bailey, Ann Marie; Testa, Charles A.; Scherman, Hataichanok; Crick, Dean C.

    2005-01-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl dipho...

  4. Mechanism and inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

    Murkin, Andrew S; Manning, Kathryn A; Kholodar, Svetlana A

    2014-12-01

    The non-mevalonate or 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is responsible for generating isoprenoid precursors in plants, protozoa, and bacteria. Because this pathway is absent in humans, its enzymes represent potential targets for the development of herbicides and antibiotics. 1-Deoxy-d-xylulose (DXP) reductoisomerase (DXR) is a particularly attractive target that catalyzes the pathway's first committed step: the sequential isomerization and NADPH-dependent reduction of DXP to MEP. This article provides a comprehensive review of the mechanistic and structural investigations on DXR, including its discovery and validation as a drug target, elucidation of its chemical and kinetic mechanisms, characterization of inhibition by the natural antibiotic fosmidomycin, and identification of structural features that provide the molecular basis for inhibition of and catalysis. PMID:24998420

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

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

    2016-09-01

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

  6. Design of Potential Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)—Evidence of a Novel Binding Mode

    San Jose, Géraldine; Jackson, Emily R.; Uh, Eugene; Johny, Chinchu; Haymond, Amanda; Lundberg, Lindsay; Pinkham, Chelsea; Kehn-Hall, Kylene; Boshoff, Helena I.; Couch, Robin D.; Dowd, Cynthia S.

    2013-01-01

    In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. ...

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

    Juan Liu; Yanhong Xu; Liang Liang; Jianhe Wei

    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 ($\\alpha$-guaiene, $\\alpha$-humulene and $\\delta$-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.

  8. Design of Potential Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode.

    San Jose, Géraldine; Jackson, Emily R; Uh, Eugene; Johny, Chinchu; Haymond, Amanda; Lundberg, Lindsay; Pinkham, Chelsea; Kehn-Hall, Kylene; Boshoff, Helena I; Couch, Robin D; Dowd, Cynthia S

    2013-07-01

    In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 µM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents. PMID:23914289

  9. Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA method.

    Ruiyang Zou

    Full Text Available The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP pathway were assembled using multiple DNA modules. A plasmid (21.6 kb containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10(3 cfu/ µg input DNA within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC, an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.

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

    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.

  11. 烟草5-磷酸脱氧木酮糖还原异构酶基因(dxr)的克隆和表达分析%Cloning and Expression Analysis of 1-deoxy-D-xylulose-5-phosphate Reductoisomerases Gene (dxr) in Nicotina tabacum

    朱晓宇; 王景; 赵二卫; 姚姗姗; 崔红

    2011-01-01

    以烟草(Nicotiana tabacum)栽培品种K326为材料,采用RT-PCR技术,克隆了萜类代谢关键酶烟草5-磷酸脱氧木酮糖还原异构酶(dxr)的cDNA片段.该基因编码区长1422 bp,编码473个氨基酸残基.利用Clustal W(1.82)和Bioedit软件,对烟草与番茄(Lycopersicon esculentum)、长春花(Catharanthus roseus)、金鱼草(A ntirrhinum majus)、薄荷(Mentha piperita)、玉米(Zea mays)、拟南芥(A rabidopsis thaliana)、念珠藻(Nostoc sp.)等物种dar基因的同源性进行分析,其氨基酸同源性分别达到93.6%、87.9%、86.3%、84.6%、84.2%、82.9%和53.5%.原核表达结果证明,该基因编码蛋白的分子量约为50 kD,与氨基酸序列估算相符合.组织表达特异性分析表明,dxr基因在烟草组织中的表达强弱为花>叶>茎>腺毛>种子>根,在花和叶片中的表达量占优势.该结果对烟草萜类代谢的分子调控和品质改良具有重要的参考价值.%Isoprenoid biosynthesis via mvalonate-independent pathway is very important to tobacco resistance and leaf quality. 1-deoxy-D-Xylulose-5-phosphate Reductoisomerases (dxr) is a key enzyme in biosynthesis of isopentenyl diphosphate, which is the precusor for monoterpenoid, diterpenoid and tetratepenoid compounds. To regulate the terpenoid metabolism pathway for tobacco improvement, some important genes such as dxr should be studied firstly. In this paper, dxr gene was cloned successfully from tobacco (Nicotiana tabacum) cultivar K326 leaf by RT-PCR. The cDNA code region was 1 422 bp long and encoding 437 amino acids. Sequence analysis by Clustal W declared that this fragment was highly homologous to dxr gene of other species. It shared 93.6% amino acid homologous to Lycopersicon esculentum, 87.9% to Catharanthus roseus, 86.3% to Antirrhinum majus,84.6% to Mentha piperita, 84.2% to Zea mays, 82.9% to Arabidopsis thaliana, and 53.5% to Nostoc sp.PCC7120. The expression vector pET21b-dxr was constructed and expressed in

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

    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 ...

  13. The Effect of Transient HMG-CoA Reductase and 1-Deoxy-D-Xylulose-5-Phosphate Synthase Overexpression on Terpene Production in Transgenic Tomato Fruits

    Gentry, Scott A; Gutensohn, Michael; Henry, Laura; Dudareva, Natalia

    2014-01-01

    Isoprenoids are secondary metabolites that control numerous plant functions including signaling, growth, photosynthesis, and membrane structure. The bioengineering of isoprenoid synthesis could produce plants with a variety of beneficial traits. Plants form isoprenoids using two different pathways, the mevalonate (MVA) pathway and the methylerithritol phosphate (MEP) pathway, which cooperate via metabolic cross-talk. Transgenic tomato lines expressing both the plastidic and cytosolic forms of...

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

    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.

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

    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 reso...

  16. Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway

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

    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 pro...

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

    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.

  18. Arabidopsis CDS blastp result: AK106750 [KOME

    Full Text Available AK106750 002-115-C09 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  19. Arabidopsis CDS blastp result: AK104851 [KOME

    Full Text Available AK104851 001-043-A10 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  20. Arabidopsis CDS blastp result: AK100909 [KOME

    Full Text Available AK100909 J023132G24 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylul ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  1. Arabidopsis CDS blastp result: AK058950 [KOME

    Full Text Available AK058950 001-020-A07 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  2. Arabidopsis CDS blastp result: AK059821 [KOME

    Full Text Available AK059821 006-205-D11 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  3. Arabidopsis CDS blastp result: AK064944 [KOME

    Full Text Available AK064944 J013000P14 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylul ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  4. AcEST: DK948936 [AcEST

    Full Text Available GYGT+VQN L Sbjct: 582 IPIEVGRGRILLEGTEVALLGYGTMVQNCL 611 >tr|Q84ML9|Q84ML9_ANDPA 1-deoxy-D-xylulose-5-phosphate synthase OS=Andrograp...his paniculata GN=DXS1 PE=2 SV=1 Length = 691 Score = 38

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

    Lundgren Anneli

    2011-03-01

    Full Text Available Abstract 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

  6. Predicted essential proteins ofPlasmodium falciparum for potential drug targets

    Qing-Feng He; Li Deng; Qin-Ying Xu; Zheng Shao

    2012-01-01

    ABSTRACT Objective:To identify novel drug targets for treatment ofPlasmodium falciparum.Methods:LocalBLASTP were used to find the proteins non-homologous to human essential proteins as novel drug targets. Functional domains of novel drug targets were identified by InterPro and Pfam,3D structures of potential drug targets were predicated by theSWISS-MODELworkspace. Ligands and ligand-binding sites of the proteins were searched byEf-seek.Results:Three essential proteins were identified that might be considered as potential drug targets.AAN37254.1 belonged to1-deoxy-D-xylulose5-phosphate reductoisomerase,CAD50499.1 belonged to chorismate synthase,CAD51220.1 belonged toFAD binging3 family, but the function of CAD51220.1 was unknown. The3D structures, ligands and ligand-binding sites ofAAN37254.1 andCAD50499.1 were successfully predicated.Conclusions:Two of these potential drug targets are key enzymes in2-C-methyl-d-erythritol4-phosphate pathway and shikimate pathway, which are absent in humans, so these two essential proteins are good potential drug targets. The function and3D structures ofCAD50499.1 is still unknown, it still need further study.

  7. Enhanced Production of Bioactive Isoprenoid Compounds from Cell Suspension Cultures of Artemisia annua L. Using β-Cyclodextrins

    Francesca Rizzello

    2014-10-01

    Full Text Available Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold, Q9 (3-fold and Q10 (2.5-fold. Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

  8. Enhanced production of bioactive isoprenoid compounds from cell suspension cultures of Artemisia annua L. using β-cyclodextrins.

    Rizzello, Francesca; De Paolis, Angelo; Durante, Miriana; Blando, Federica; Mita, Giovanni; Caretto, Sofia

    2014-01-01

    Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules. PMID:25338048

  9. Enhanced Production of Bioactive Isoprenoid Compounds from Cell Suspension Cultures of Artemisia annua L. Using β-Cyclodextrins

    Rizzello, Francesca; De Paolis, Angelo; Durante, Miriana; Blando, Federica; Mita, Giovanni; Caretto, Sofia

    2014-01-01

    Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules. PMID:25338048

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

    Jordi Perez-Gil

    Full Text Available 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.

  11. Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

    Smith, Jessica M.; Warrington, Nicole V.; Vierling, Ryan J.; Kuhn, Misty L.; Anderson, Wayne F; Koppisch, Andrew T.; Freel Meyers, Caren L.

    2013-01-01

    The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhi...

  12. Ketol-acid reductoisomerase enzymes and methods of use

    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.

  13. Ketol-acid reductoisomerase enzymes and methods of use

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

    2016-07-12

    Provided herein are polypeptides having ketol-acid 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.

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

    Mei Han

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

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

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

    2013-01-01

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

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

    ... 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 ...

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

    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. PMID:25926480

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

    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. PMID:18211549

  19. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    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...

  20. De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy.

    Masini, T.; Pilger, J.; Kroezen, B.; Illarionov, B.; Lottmann, P.; Fischer, M.; Griesinger, C.; Hirsch, A.

    2014-01-01

    We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans D...

  1. Purification and characterization of ribitol-5-phosphate and xylitol-5-phosphate dehydrogenases from strains of Lactobacillus casei.

    Hausman, S Z; London, J

    1987-01-01

    A simple three-step procedure is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol...

  2. Lipophilic prodrugs of FR900098 are antimicrobial against Francisella novicida in vivo and in vitro and show GlpT independent efficacy.

    Elizabeth S McKenney

    Full Text Available Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase. MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC(50=230 nM. Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed "compound 1," was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad

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

    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…

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

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

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive dru...

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

    Nichols, Trent W; Gaiteri, Christopher

    2014-12-01

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

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

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

    2015-04-01

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

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

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

    2016-05-01

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

  8. Molecular design, synthesis and biological activities of amidines as new ketol-acid reductoisomerase inhibitors

    Bao Lei Wang; Yong Hong Li; Jian Guo Wang; Yi Ma; Zheng Ming Li

    2008-01-01

    Diamidine (A) was identified in our in vitro bio-assay as a possible inhibitor of ketol-acid reductoisomerase (KARI) from the ACD database search based on the known three-dimensional crystal structure of KARI. An investigation on interaction of A on KARI active sites, led to the design and synthesis of 15 novel monoamidines. Some of those showed better biological activity than A on rice KARI (in vitro) and in greenhouse herbicidal tests (in vivo). The structure-biological activity relationship was investigated, which provides valuable information to further study of potential KARI inhibitors.

  9. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. PMID:23954075

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

    The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures. 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

  11. Preparation and Purification of Xylitol-5-Phosphate from a Cell Extract of Lactobacillus casei Cl-16

    Trahan, L.; Néron, S.; Bareil, M.

    1988-01-01

    A simple procedure which yields pure xylitol-5-phosphate is described. A cell extract of Lactobacillus casei Cl-16 from a 6-liter culture was used to synthesize up to 70 mg of xylitol-5-phosphate overnight from xylitol and phosphoenolpyruvate via a xylitol phosphoenolpyruvate:phosphotransferase system with a 53% yield. Centrifugation, filtration, precipitation as a barium salt, and ion-exchange batch chromatography permitted recovery of nearly 90% of the phosphorylated product synthesized. Th...

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

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

    1984-02-14

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

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

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

    2016-08-01

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

  14. LuxS-independent formation of AI-2 from ribulose-5-phosphate

    Hardie Kim R

    2008-06-01

    Full Text Available Abstract Background In many bacteria, the signal molecule AI-2 is generated from its precursor S-ribosyl-L-homocysteine in a reaction catalysed by the enzyme LuxS. However, generation of AI-2-like activity has also been reported for organisms lacking the luxS gene and the existence of alternative pathways for AI-2 formation in Escherichia coli has recently been predicted by stochastic modelling. Here, we investigate the possibility that spontaneous conversion of ribulose-5-phosphate could be responsible for AI-2 generation in the absence of luxS. Results Buffered solutions of ribulose-5-phosphate, but not ribose-5-phosphate, were found to contain high levels of AI-2 activity following incubation at concentrations similar to those reported in vivo. To test whether this process contributes to AI-2 formation by bacterial cells in vivo, an improved Vibrio harveyi bioassay was used. In agreement with previous studies, culture supernatants of E. coli and Staphylococcus aureus luxS mutants were found not to contain detectable levels of AI-2 activity. However, low activities were detected in an E. coli pgi-eda-edd-luxS mutant, a strain which degrades glucose entirely via the oxidative pentose phosphate pathway, with ribulose-5-phosphate as an obligatory intermediate. Conclusion Our results suggest that LuxS-independent formation of AI-2, via spontaneous conversion of ribulose-5-phosphate, may indeed occur in vivo. It does not contribute to AI-2 formation in wildtype E. coli and S. aureus under the conditions tested, but may be responsible for the AI-2-like activities reported for other organisms lacking the luxS gene.

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

    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. PMID:26780217

  16. Positive outcome following early diagnosis and treatment of pyridoxal-5'-phosphate oxidase deficiency: a case report.

    Porri, Stephanie; Fluss, Joel; Plecko, Barbara; Paschke, Eduard; Korff, Christian M; Kern, Ilse

    2014-02-01

    Pyridoxal-5'-phosphate oxidase (PNPO) deficiency is a rare autosomal recessive, vitamin-responsive metabolic disorder causing refractory neonatal seizures that respond to the administration of pyridoxal-5'-phosphate (PLP). There are currently few case studies that have documented the functional outcome in PNPO deficiency, which remains poor in the majority of cases. We present the case of a male infant born at 35 weeks gestation who promptly responded to oral administration of PLP, following resistance to common anticonvulsive therapy and to a pyridoxine trial. Neurological outcome at 21 months is favorable and illustrates the importance of standardized vitamin trials in the acute setting of "therapy-resistant" neonatal seizures. Early recognition of PNPO deficiency and appropriate intervention might be associated with a more favorable outcome than initially considered. PMID:24297574

  17. Phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ), a lipid signalling enigma.

    Giudici, Maria-Luisa; Clarke, Jonathan H; Irvine, Robin F

    2016-05-01

    The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an important family of enzymes, whose physiological roles are being teased out by a variety of means. Phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) is especially intriguing as its in vitro activity is very low. Here we review what is known about this enzyme and discuss some recent advances towards an understanding of its physiology. Additionally, the effects of the ATP-competitive inhibitor I-OMe Tyrphostin AG-538 on all three mammalian PI5P4Ks was explored, including two PI5P4Kγ mutants with altered ATP- or PI5P-binding sites. The results suggest a strategy for targeting non-ATP binding sites on inositol lipid kinases. PMID:26710750

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

    Leibly David J

    2011-10-01

    Full Text Available Abstract 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.

  19. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  20. Long-term effect of magnesium pyridoxal 5-phosphate glutamate in rabbits developing hypercholesterolemia.

    Panagiotopoulos, T; Ketelsen, U P; Schmidt, A; Heuck, C C

    1986-08-01

    36 male rabbits were fed with a diet enriched with 20 g cholesterol/kg for a period of 8 weeks under a strict daily dietary control. Magnesium pyridoxal 5-phosphate glutamate (MPPG, Sedalipid) was supplemented to the diet at different quantities. Hypercholesterolemia developed later and less pronounced in animals receiving a medium or high dose of MPPG. Microscopical analysis indicated a protective effect of MPPG on calcium deposition in the aorta. The fatty acid pattern in serum showed only minor differences and was unchanged in liver extracts in animals supplemented with MPPG. This observation suggest that MPPG may act on the mechanism of cholesterol absorption in the intestine. PMID:3778558

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

    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.

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

    The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[α-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 370C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli, the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken

  3. Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans

    Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses. Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni2+-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing

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

    Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 m-1 s-1 at 20C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence, two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. The reactive sulfhydryl groups are likely to be those generated by reduction of a disulfide during activation. 20 references, 3 figures, 2 tables

  5. Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy.

    Heintze, Jacob; Costa, Joana R; Weber, Melanie; Ketteler, Robin

    2016-09-01

    Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling. PMID:27328773

  6. Binding and uptake of 125iodine-labelled, oxidized low density lipoprotein by macrophages: Comparison of the effects of α-tocopherol, probucol, pyridoxal-5'-phosphate and magnesium-pyridoxal-5'-phosphate-glutamate

    Specific and unspecific binding and uptake (internalization) by macrophages of 125iodine-labelled, copper-oxidized human low density lipoprotein is differently influenced by the anti-oxidants α-tocopherol (α-Toc), probucol (Prob), pyridoxal-5'-phosphate (PP) and the magnesium-pyridoxal-5'-phosphate glutamate complex (MPPG). Binding as well as internalization, mediated by the so-called 'scavenger receptor' is lower in the presence of MPPG whereas both specific binding and internalization are enhanced. The comparison of the effects in vitro allows a rating of the potentially anti-atherogenic and thus protective effects of the tested substances as follows: MPPG>PP>α-Toc>Prob. (orig.)

  7. Binding and uptake of 125iodine-labelled, oxidized low density lipoprotein by macrophages: comparison of the effects of alpha-tocopherol, probucol, pyridoxal-5'-phosphate and magnesium-pyridoxal-5'-phosphate-glutamate.

    Selmer, D; Senekowitsch-Schmidtke, R; Schneider, W; Elstner, E F

    1997-01-01

    Specific and unspecific binding and uptake (internalization) by macrophages of 125iodine-labelled, copper-oxidized human low density lipoprotein is differently influenced by the anti-oxidants alpha-tocopherol (alpha-Toc), probucol (Prob), pyridoxal-5'-phosphate (PP) and the magnesium-pyridoxal-5'-phosphate glutamate complex (MPPG). Binding as well as internalization, mediated by the so-called "scavenger receptor" is lower in the presence of MPPG whereas both specific binding and internalization are enhanced. The comparison of the effects in vitro allows a rating of the potentially anti-atherogenic and thus protective effects of the tested substances as follows: MPPG > PP > alpha-Toc > Prob. PMID:9090072

  8. Binding and uptake of {sup 125}iodine-labelled, oxidized low density lipoprotein by macrophages: Comparison of the effects of {alpha}-tocopherol, probucol, pyridoxal-5`-phosphate and magnesium-pyridoxal-5`-phosphate-glutamate

    Selmer, D. [Technische Univ. Muenchen, Freising-Weihenstephan (Germany). Lehrstuhl fuer Phytopathologie; Senekowitsch-Schmidtke, R. [Technische Univ. Muenchen (Germany). Nuklearmedizinische Klinik; Schneider, W. [Steigerwald Arzneimittel, Darmstadt (Germany); Elstner, E.F. [Technische Univ. Muenchen, Freising-Weihenstephan (Germany). Lehrstuhl fuer Phytopathologie

    1997-01-01

    Specific and unspecific binding and uptake (internalization) by macrophages of {sup 125}iodine-labelled, copper-oxidized human low density lipoprotein is differently influenced by the anti-oxidants {alpha}-tocopherol ({alpha}-Toc), probucol (Prob), pyridoxal-5`-phosphate (PP) and the magnesium-pyridoxal-5`-phosphate glutamate complex (MPPG). Binding as well as internalization, mediated by the so-called `scavenger receptor` is lower in the presence of MPPG whereas both specific binding and internalization are enhanced. The comparison of the effects in vitro allows a rating of the potentially anti-atherogenic and thus protective effects of the tested substances as follows: MPPG>PP>{alpha}-Toc>Prob. (orig.)

  9. Inhibitor design for ribonuclease A: the binding of two 5'-phosphate uridine analogues.

    Tsirkone, Vicky G; Dossi, Kyriaki; Drakou, Christina; Zographos, Spyros E; Kontou, Maria; Leonidas, Demetres D

    2009-07-01

    In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5'-phosphate (U5P) and uridine 5'-diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with K(i) values of 4.0 and 0.65 mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purine-binding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily. PMID:19574636

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

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

    2016-04-01

    2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/β)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and β8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility. PMID:27033207

  11. Magnesium pyridoxal 5-phosphate glutamate reduces hyperlipidaemia in patients with chronic renal insufficiency.

    Kirsten, R; Heintz, B; Nelson, K; Sieberth, H G; Oremek, G; Hasford, J; Speck, U

    1988-01-01

    Chronic renal insufficiency is often accompanied by hyperlipidaemia and subsequent coronary heart disease. Two groups of 15 patients with serum creatinine greater than 2 mg/100 ml and serum cholesterol less than 250 mg/100 ml were given 3 x 50 mg magnesium pyridoxal 5-phosphate glutamate (MPPG) or placebo for 12 weeks in a double-blind, randomised study. Total cholesterol in the MPPG group (282.4 mg.100 ml-1) was lower than in the placebo group (354.3 mg.100 ml-1) after 12 weeks of treatment. Triglycerides in the MPPG group were 265.1 mg.100 ml-1 compared to 361.9 mg.100 ml-1. After 12 weeks on MPPG the LDL/HDL ratio of 3.56 was lower than in the placebo group-6.83. Side effects in the MPPG group were similar to those in the placebo group. Thus, MPPG was an effective antihyperlipidaemic agent in patients with renal insufficiency. PMID:3383985

  12. Initiation sites for human DNA replication at a putative ribulose-5-phosphate 3-epimerase gene

    Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes. Each replicon contains an initiation, or origin, site, at which DNA synthesis begins. However, very little information is known about the nature and positioning of these initiation sites along human chromosomes. We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae. This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE). In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene. By applying this analysis to a 1-1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2. The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene

  13. Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures

    Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response

  14. Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.

    Mohini S Ghatge

    Full Text Available Pyridoxal 5'-phosphate (PLP is a cofactor for dozens of B(6 requiring enzymes. PLP reacts with apo-B(6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

  15. Effects of Gibberellic Acid on Primary Terpenoids and △9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

    Hakimeh Mansouri; Zahra Asrar; Mitra Mehrabani

    2009-01-01

    Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration,and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP)pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA3) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA3 caused a decrease in DXS activity in both sexes that it wasaccompanied by a decrease in chlorophylls, carotenoids and △9-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA3 showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants.The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA3 can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.

  16. Enhanced Diterpene Tanshinone Accumulation and Bioactivity of Transgenic Salvia miltiorrhiza Hairy Roots by Pathway Engineering.

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Li, Leilei; Huang, Shengxiong; Zhang, Tong; Wang, Huizhong; Kai, Guoyin

    2016-03-30

    Tanshinones are health-promoting diterpenoids found in Salvia miltiorrhiza and have wide applications. Here, SmGGPPS (geranylgeranyl diphosphate synthase) and SmDXSII (1-deoxy-d-xylulose-5-phosphate synthase) were introduced into hairy roots of S. miltiorrhiza. Overexpression of SmGGPPS and SmDXSII in hairy roots produces higher levels of tanshinone than control and single-gene transformed lines; tanshinone production in the double-gene transformed line GDII10 reached 12.93 mg/g dry weight, which is the highest tanshinone content that has been achieved through genetic engineering. Furthermore, transgenic hairy root lines showed higher antioxidant and antitumor activities than control lines. In addition, contents of chlorophylls, carotenoids, indoleacetic acid, and gibberellins were significantly elevated in transgenic Arabidopsis thaliana plants. These results demonstrate a promising method to improve the production of diterpenoids including tanshinone as well as other natural plastid-derived isoprenoids in plants by genetic manipulation of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. PMID:26753746

  17. Effect of magnesium pyridoxal 5-phosphate glutamate on vascular reactivity in experimental hypercholesterolemia.

    Khayyal, M T; Khayyal, M A; Sharaf, H M; el-Sherbeeny, M; Okpanyi, S N; Schneider, W

    1998-01-01

    Hypercholesterolemia is known to affect the responsiveness of various blood vessels to endogenous and to exogenous vasoactive agents. Of particular interest is the increased responsiveness to vasoconstrictors, e.g., 5-hydroxy tryptamine and noradrenaline, and the decreased reactivity towards vasodilators, e.g., acetylcholine. This, together with the development of arteriosclerosis, could play an important role in the progression of many vascular complications, such as hypertension and coronary heart disease. Magnesium pyridoxal 5-phosphate glutamate (MPPG) has been shown to effectively reduce serum lipids in animals and in man, and to retard the progression of atherosclerotic lesions in experimental animals. It was therefore considered of interest to investigate the reactivity of both the aorta and the renal artery to different vasoactive substances in hypercholesterolemic rabbits under the influence of MPPG as well as the effect of such substances on the blood pressure of the anesthetized animals. The rabbits were fed a high cholesterol diet for 2 months, followed by MPPG for 1 month, while keeping the rabbits on the same diet. One batch of animals was used for blood pressure recording and testing drug effects, and another was used for testing the responsiveness of their aortae and renal arteries to the different mediators. In hypercholesterolemic rabbits, treatment with MPPG tended to normalize the increased responsiveness of the blood pressure to the vasoconstrictors: noradrenaline and angiotensin and the diminished sensitivity to histamine and acetylcholine. For the isolated arteries, however, MPPG did not significantly affect the responses to noradrenaline nor potassium chloride, but tended to normalize responses to clonidine and acetylcholine. It could be concluded from the present findings that the high cholesterol diet induces changes in vascular reactivity which are possibly related to endothelial and/or receptor sensitivity changes. Treatment with MPPG

  18. On the mechanism of Escherichia coli pyridoxal kinase inhibition by pyridoxal and pyridoxal 5'-phosphate.

    di Salvo, Martino Luigi; Nogués, Isabel; Parroni, Alessia; Tramonti, Angela; Milano, Teresa; Pascarella, Stefano; Contestabile, Roberto

    2015-09-01

    Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. PMID:25655354

  19. Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve

    Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl2, 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution

  20. Ribose-5-Phosphate Isomerase Deficiency: New Inborn Error in the Pentose Phosphate Pathway Associated with a Slowly Progressive Leukoencephalopathy

    Huck, Jojanneke H. J.; Verhoeven, Nanda M; Struys, Eduard A.; Salomons, Gajja S; Jakobs, Cornelis; van der Knaap, Marjo S.

    2004-01-01

    The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated...

  1. Markedly increased circulating pyridoxal-5'-phosphate levels in hypophosphatasia. Alkaline phosphatase acts in vitamin B6 metabolism.

    Whyte, M P; Mahuren, J D; Vrabel, L A; Coburn, S P

    1985-01-01

    Markedly increased circulating concentrations of pyridoxal-5'-phosphate (PLP) were found in each of 14 patients representing all clinical forms of hypophosphatasia, an inborn error characterized by deficient activity of the tissue nonspecific (bone/liver/kidney) isoenzyme of alkaline phosphatase (AP). The mean PLP concentration in plasma was 1174 nM (range, 214-3839 nM) in the patients and 57 +/- 26 nM (mean +/- SD) in 38 control subjects. In four affected children, urinary excretion of the P...

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

    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. PMID:27297548

  3. Preliminary X-ray crystallographic analysis of the d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis

    The expression, purification, preliminary crystallization and crystallographic analysis of phosphoketolase from L. lactis ssp. lactis (strain IL 1403) are reported. Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon–carbon bond of the specific substrate d-xylulose 5-phosphate (or d-fructose 6-phosphate) to give acetyl phosphate and d-glyceraldehyde 3-phosphate (or d-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P21. Diffraction data were obtained to a resolution of 2.2 Å

  4. Ribose-5-Phosphate Isomerase Deficiency: New Inborn Error in the Pentose Phosphate Pathway Associated with a Slowly Progressive Leukoencephalopathy

    Huck, Jojanneke H. J.; Verhoeven, Nanda M.; Struys, Eduard A.; Salomons, Gajja S.; Jakobs, Cornelis; van der Knaap, Marjo S.

    2004-01-01

    The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene–sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders. PMID:14988808

  5. Mechanism of the inhibition of mutagenicity of a benzo[a]pyrene 7,8-diol 9,10-epoxide by riboflavin 5'-phosphate.

    Wood, A.W.; Sayer, J M; Newmark, H L; Yagi, H.; Michaud, D P; Jerina, D M; Conney, A H

    1982-01-01

    Riboflavin 5'-phosphate (flavin mononucleotide; FMN) inhibits the mutagenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P diol epoxide), the only known ultimate carcinogenic metabolite of benzo[a]pyrene. Coincubation of 10, 25, and 50 nmol of FMN with strain TA100 of histidine-dependent Salmonella typhimurium inhibits the mutagenicity of 0.05 nmol of the diol epoxide by 50, 70, and 90%, respectively. Ribose 5-phosphate and riboflavin sh...

  6. Radiation damage to nucleosides and nucleotides. I. An ESR study of uridine-5'-phosphate.2Na+ single crystals

    Single crystals of the disodium salt of uridine-5'-phosphate were irradiated by 4.0-MeV electrons and studied by electron spin resonance spectroscopy in the temperature range 77 to 310 K. At 77 K two resonances were observed. The dominating resonance (I) is ascribed to the electron adduct (anion) radical of the uracil base. The main hyperfine interaction observed is a coupling between the unpaired electron and the proton bonded to C6. The other resonance (II) exhibits a large g-value anisotropy and two almost isotropic doublet couplings. This resonance is probably due to a secondary alkoxy-type radical formed by a net loss of hydrogen from O3' in the sugar moiety. At 270 K three resonances were observed. One (III) is due to a radical formed by hydrogen abstraction from C/sub 5'/ in the sugar moiety, whereas the other two (IV,V) are probably hydrogen addition radicals at the uracil base. Radical IV is the dihydrouracil-5-yl radical formed by a net hydrogen addition to the C6 position, whereas radical V tentatively is ascribed to the 6-yl radical. The light-induced conversion V → IV took place at 270 K, whereas the reverse heat-induced process was not observed in the temperature range studied. Preliminary annealing studies of crystals irradiated at 77 K suggest that the radical reaction I → IV takes place at approximately 130 K and the reaction II → III at 150 K

  7. Large-Scale Domain Motions and Pyridoxal-5'-Phosphate Assisted Radical Catalysis in Coenzyme B12-Dependent Aminomutases

    Amarendra Nath Maity

    2014-02-01

    Full Text Available Lysine 5,6-aminomutase (5,6-LAM and ornithine 4,5-aminomutase (4,5-OAM are two of the rare enzymes that use assistance of two vitamins as cofactors. These enzymes employ radical generating capability of coenzyme B12 (5'-deoxyadenosylcobalamin, dAdoCbl and ability of pyridoxal-5'-phosphate (PLP, vitamin B6 to stabilize high-energy intermediates for performing challenging 1,2-amino rearrangements between adjacent carbons. A large-scale domain movement is required for interconversion between the catalytically inactive open form and the catalytically active closed form. In spite of all the similarities, these enzymes differ in substrate specificities. 4,5-OAM is highly specific for D-ornithine as a substrate while 5,6-LAM can accept D-lysine and L-β-lysine. This review focuses on recent computational, spectroscopic and structural studies of these enzymes and their implications on the related enzymes. Additionally, we also discuss the potential biosynthetic application of 5,6-LAM.

  8. Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

    Tim Soderberg

    2005-01-01

    Full Text Available A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP, the ribulose monophosphate (RuMP pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium produce ribose-5-phosphate via the nonoxidative PPP (NOPPP, whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1 lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP, the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii probably does not synthesize aromatic amino acids at all.

  9. Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization.

    Sun, Fangfang; Zhang, Xiao-Zhou; Myung, Suwan; Zhang, Y-H Percival

    2012-04-01

    The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs. PMID:22333529

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

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

    2016-04-01

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

  11. Studies on the increase of capillary permeability in rat skin under the action of pyridoxal 5' phosphate

    The activity of pyridoxal 5'-phosphate (PLP) is described on the vascular permeability response, measured in the abdominal wall of rats from the amount of extravased Evans blue labelled with radioactive iodine 125 or 131. The PLP effect is related to histamine release as it has been showed by tha use of antihistaminics. An attempt has been made in order to correlate structure and biological activity by using PLP analogs. The intact molecule of PLP seems to be the proper active substance. The critical role of calcium in histamine release is discussed in relation to our observations. In the presence of high concentrations of calcium and lantanium, PLP fails to increase the vascular permeability; magnesium does not show any influence. The calcium mobilization produced by theophylline results in inhibition of the response. The course of the reaction between PLP and histamine in vitro was followed; the synthetic cyclic product is deprived of activity and does not interfere with the intrinsic effects of PLP and histamine. (Author)

  12. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

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

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

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

  14. Method of preparing highly active and thermostable preparations of liver uridin-kinase usable for enzymic synthesis of radioactive nucleoside-5'-phosphates

    A method is described of preparing a high-activity uridine kinase for the enzymic synthesis of radioactive nucleoside-5m-phosphates of the pyrimidine series. The preparation is separated from male rat liver after intraperitoneal application of 5'-azacytidine. Examples are given showing detailed procedures for the conversion of uridine and 6-azauridine to the corresponding 5'-phosphates. (L.K.)

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

    Luis Enrique Gutierrez-Millan

    2009-02-01

    Full Text Available 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 photoautotrophicgrowth 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 conversionof 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.

  16. A candidate gene association study on muscat flavor in grapevine (Vitis vinifera L.

    Boursiquot Jean-Michel

    2010-11-01

    Full Text Available Abstract Background The sweet, floral flavor typical of Muscat varieties (Muscats, due to high levels of monoterpenoids (geraniol, linalool and nerol, is highly distinct and has been greatly appreciated both in table grapes and in wine since ancient times. Muscat flavor determination in grape (Vitis vinifera L. has up to now been studied by evaluating monoterpenoid levels through QTL analysis. These studies have revealed co-localization of 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS with the major QTL positioned on chromosome 5. Results We resequenced VvDXS in an ad hoc association population of 148 grape varieties, which included muscat-flavored, aromatic and neutral accessions as well as muscat-like aromatic mutants and non-aromatic offsprings of Muscats. Gene nucleotide diversity and intragenic linkage disequilibrium (LD were evaluated. Structured association analysis revealed three SNPs in moderate LD to be significantly associated with muscat-flavored varieties. We identified a putative causal SNP responsible for a predicted non-neutral substitution and we discuss its possible implications for flavor metabolism. Network analysis revealed a major star-shaped cluster of reconstructed haplotypes unique to muscat-flavored varieties. Moreover, muscat-like aromatic mutants displayed unique non-synonymous mutations near the mutated site of Muscat genotypes. Conclusions This study is a crucial step forward in understanding the genetic regulation of muscat flavor in grapevine and it also sheds light on the domestication history of Muscats. VvDXS appears to be a possible human-selected locus in grapevine domestication and post-domestication. The putative causal SNP identified in Muscat varieties as well as the unique mutations identifying the muscat-like aromatic mutants under study may be immediately applied in marker-assisted breeding programs aimed at enhancing fragrance and aroma complexity respectively in table grape and wine cultivars.

  17. Competence of Thiamin Diphosphate-Dependent Enzymes with 2'-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin.

    Nemeria, Natalia S; Shome, Brateen; DeColli, Alicia A; Heflin, Kathryn; Begley, Tadhg P; Meyers, Caren Freel; Jordan, Frank

    2016-02-23

    Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2'-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and Escherichia coli pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the E. coli 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), and the human and E. coli 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to E. coli PDHc (6-11%) and DXPS (9-14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP. PMID:26813608

  18. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

    Zou Ruiyang

    2011-04-01

    Full Text Available Abstract Background Accurate interpretation of quantitative PCR (qPCR data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.

  19. Reduced neuronal expression of ribose-5-phosphate isomerase enhances tolerance to oxidative stress, extends lifespan, and attenuates polyglutamine toxicity in Drosophila

    Wang, Ching-Tzu; Chen, Yi-Chun; Wang, Yi-Yun; Huang, Ming-Hao; Yen, Tzu-Li; Li, Hsun; Liang, Cyong-Jhih; Sang, Tzu-Kang; Cho, Si-Chih; Yuh, Chiou-Hwa; Wang, Chao-Yung; Brummel, Theodore J.; Wang, Horng-Dar

    2011-01-01

    Aging and age-related diseases can be viewed as the result of the lifelong accumulation of stress insults. The identification of mutant strains and genes which are responsive to stress and can alter longevity profiles provides new therapeutic targets for age-related diseases. Here we reported that a Drosophila strain with reduced expression of ribose-5-phosphate isomerase (rpi), EP2456, exhibits increased resistance to oxidative stress and enhanced lifespan. In addition, the strain also displ...

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

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

    1989-01-01

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

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

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (β/α)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 Zn2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn2+ and inactive apoenzyme cannot be prepared, the affinity for Zn2+ is decreased by alanine substitutions for the two histidine residues that coordinate the Zn2+ ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn2+. 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 Zn2+ 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 Zn2+ and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif

  2. Stability of pyridoxal-5-phosphate semicarbazone: applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status.

    Ubbink, J B; Serfontein, W J; de Villiers, L S

    1985-08-01

    The determination of pyridoxal-5-phosphate (PLP) and pyridoxal (PL) in plasma requires the availability of dark room facilities, due to the photosensitivity of these vitamin B6 vitamers. The fact that the semicarbazone forms of PL and PLP are more strongly fluorescent than the underivatized B6 vitamers has been exploited in plasma analyses, but it was not previously realised that these semicarbazone forms are also very stable even under conditions that lead to rapid decomposition of free PL and PLP. The stabilisation of PLP and PL obtained in this manner is sufficient and fully adequate to meet the practical requirements of clinical field studies. We report a high-performance liquid chromatographic method for plasma PLP and PL determinations based on precolumn semicarbazone formation and fluorescence detection. The method is sensitive enough for quantitative plasma PLP determinations even in B6-deficient patients. PMID:4055950

  3. Preparation of riboflavin 5' - phosphate sodium from crude riboflavin%利用核黄素粗品制备核黄素5'-磷酸钠

    魏转; 周强; 孙文敬; 崔凤杰; 张铎; 余泗莲

    2011-01-01

    The objective of the present study was to investigate the feasibility of preparing riboflavin 5' - phosphate sodium from crude riboflavin. Riboflavin 5'-phosphate sodium was obtained with the yield of 68. 5% by reacting one mole of crude riboflavin with four moles of POC13 for 2 h at 35℃ , hydrolyzing the resulting mixture, and neutralizing with NaOH solution. The quality of obtained product met the requirements of US FCC (7 ). The phosphorylation reaction conditions of riboflavin was further optimized by one-factor-at-a-time experiment design, and results were: n ( POC13 ) / n ( VB2 ) 4. 6, n (POC13) /n (H20) 1.65, reaction time 2h, and reaction temperature 35 ± 1℃. Under the above condition, the yield of riboflavin 5'-phosphate sodium increased to above 73. 5%. This method significantly decreases production cost. In conclusion, preparation of riboflavin 5'-phosphate sodium from crude riboflavin was determined to be feasible e-conomically and technically.%探索利用核黄素粗品制备核黄素5'-磷酸钠的可行性.在吡啶和乙腈的混合溶剂中,使核黄素粗品与其4倍摩尔量的三氯氧磷35℃左右下反应2h,然后水解反应混合物,再用NaOH溶液中和,能够以68.5%以上的收率得到核黄素磷酸钠,产品质量符合美国FCC (7)要求.通过单因素试验,初步优化了核黄素5'-磷酸钠粗品磷酸化反应的条件,即:n (POCl3)/n(VB2)=4.6,n(POCl3)/n(H2O)=1.65,反应时间2h,反应温度35±1℃.在优化的磷酸化反应条件下,利用核黄素粗品制备核黄素5'-磷酸钠的收率平均可达73.5%以上,能够有效降低核黄素5'-磷酸钠的生产成本.研究结果表明,利用核黄素粗品制备核黄素5'-磷酸钠无论在经济上还是在技术上均是可行的.

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

    Highlights: → We analyzed Phosphatidylinositol 5-phosphate kinase IIβ (PIPKIIβ) function in cancer. → PIPKIIβ is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. → PIPKIIβ suppresses cellular motility through E-cadherin induction in SW480 cells. → Nuclear PIP2 but not plasma membrane-localized PIP2 mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) has anti-cancer activity in several colon cancers. 1α,25(OH)2D3 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 (PIPKIIβ) but not PIPKIIα 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 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)2D3-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P2 production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)2D3. These results indicate that PIPKIIβ-mediated PI(4,5)P2 signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

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

    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

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

    Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Fujiwara, Yuki [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Yamaguchi, Hideki [Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-city, Saitama 332-0012 (Japan); Nakamura, Yoshikazu; Fukami, Kiyoko [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan)

    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.

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

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

    2010-06-01

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

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

    Matthew A Oberhardt

    2016-01-01

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

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

    A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-18F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDLP showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL except the K/sub m/ of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy. Results of this study indicate that the protein structure surrounding the coenzyme molecule, as well as the coenzyme configuration, is altered upon the binding of ligands

  10. Molecular evolution of B6 enzymes: Binding of pyridoxal-5'-phosphate and Lys41Arg substitution turn ribonuclease A into a model B6 protoenzyme

    Marra Ersilia

    2008-06-01

    Full Text Available Abstract Background The pyridoxal-5'-phosphate (PLP-dependent or vitamin B6-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B6 enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase, in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP. Results We screened the PLP adduct of wild-type RNase and two variant RNases (K7R and K41R for catalytic effects toward L- and D-amino acids. RNase(K41R-PLP, in which the cofactor is bound through an imine linkage to Lys7, qualifies for a model proto-B6 enzyme by the following criteria: (1 covalent linkage of PLP (internal aldimine; (2 catalytic activity toward amino acids that depends on formation of an imine linkage with the substrate (external aldimine; (3 adjoining binding sites for the cofactor and amino acid moiety that facilitate the transimination reaction of the internal to the external aldimine and stabilize the resulting noncovalent complex of the coenzyme-substrate adduct with the protein; (4 reaction specificity, the only detectable reactions being racemization of diverse amino acids and β-decarboxylation of L-aspartate; (5 acceleration factors for racemization and β-decarboxylation of >103 over and above that of PLP alone; (6 ribonuclease activity that is 103-fold lower than that of wild-type RNase, attenuation of a pre-existing biological activity being indispensable for the further evolution as a PLP-dependent protoenzyme. Conclusion A single amino acid substitution (Lys41Arg and covalent

  11. The influence of magnesium-pyridoxal-5'-phosphate-glutamate in comparison with probucol, alpha-tocopherol and trolox on copper-induced oxidation of human low density lipoprotein in vitro.

    Kögl, C; Schneider, W; Elstner, E F

    1994-06-15

    Low density lipoprotein (LDL) in the presence of magnesium-pyridoxal-5'-phosphate-glutamate (MPPG), pyridoxal-5'-phosphate (PP), alpha-tocopherol, probucol or trolox is more resistant against copper-induced oxidation as control-LDL in vitro. The efficiency of the drugs is: probucol > MPPG > trolox > alpha-tocopherol > PP. LDL oxidation is determined by its increasing negative surface charge, fragmentation of apolipoprotein B-100 and changes of the fatty acid content of LDL. The protection of the drugs depends on their concentration and incubation time. Different experiments point to the fact that copper-induced oxidation of LDL in vitro starts with the binding of copper at the apolipoprotein B-100, resulting in an increasing negative surface charge and fragmentation of the apolipoprotein B-100. Afterwards a decrease of LDL-bound linoleic acid (18:2) is measurable. PMID:8031313

  12. Perinatal hypophosphatasia: tissue levels of vitamin B6 are unremarkable despite markedly increased circulating concentrations of pyridoxal-5'-phosphate. Evidence for an ectoenzyme role for tissue-nonspecific alkaline phosphatase.

    Whyte, M P; Mahuren, J D; Fedde, K. N.; Cole, F. S.; McCabe, E. R.; Coburn, S P

    1988-01-01

    "Perinatal" hypophosphatasia is the most severe form of this inborn error of metabolism, which is characterized by deficient activity of the tissue-nonspecific (liver/bone/kidney) isoenzyme of alkaline phosphatase (ALP) (TNSALP). We report that autopsy tissue from three affected subjects, which was profoundly low in ALP activity, had essentially unremarkable levels of pyridoxal-5'-phosphate (PLP), pyridoxal, and total vitamin B6 content despite markedly elevated plasma PLP levels (5,800, 14,5...

  13. High yield synthesis of 14C labelled intermediates of the L-type pentose pathway: octulose mono- and biphosphates, sedoheptulose 1,7-bisphosphate and D-arabinose 5-phosphate

    Methods for the enzymic synthesis, isolation, purification and analysis of the 14C labelled intermediates that are characteristic of the L-type pentose phosphate pathway are described. These are D-glycero D-ido octulose 1,8-bisphosphate and 8-monophosphate; D-glycero D-altro octulose mono- and bisphosphates; sedoheptulose 1,7-bisphosphate and D-arabinose 5-phosphate. The authenticity of each of the 14C-labelled sugar phosphates was confirmed using a variety of chromatographic methods, enzymatic analytic methods and NMR spectroscopy. The 14C labelled compounds are used in investigations involving the search for the identity of the pentose pathway in tissues in vitro, for the measurement of L-type pathway enzyme reactions and for testing the mechanistic predictions of the L-type pathway in vitro. (author)

  14. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  15. Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system

    Posthuma, C.C.; Bader, R.; Engelmann, R.; Postma, P.W.; Hengstenberg, W.; Pouwels, P.H.

    2002-01-01

    Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described, xpkA encodes a 788-amino-acid protein with a ca

  16. Clinical analysis of vitamin B(6): determination of pyridoxal 5'-phosphate and 4-pyridoxic acid in human serum by reversed-phase high-performance liquid chromatography with chlorite postcolumn derivatization.

    Rybak, Michael E; Pfeiffer, Christine M

    2004-10-15

    A reversed-phase high-performance liquid chromatography (HPLC) method with fluorometric detection was developed for the routine determination of pyridoxal 5'-phosphate (PLP) and 4-pyridoxic acid (4-PA) in serum. Chlorite postcolumn derivatization was used to oxidize PLP to a more fluorescent carboxylic acid form. Sensitivity improved fourfold for PLP using chlorite postcolumn derivatization over traditional bisulfite postcolumn derivatization. The HPLC injection cycle was 15 min, facilitating a throughput of 60 patient samples (72 injections that included standards and quality control (QC) samples) in 18.5h. Method precision was evaluated using three serum QC pools with PLP and 4-PA concentrations of 11.5-34.8 nmol/L and 10.4-21.0 nmol/L, respectively. Within-run (n=7) repeatabilities were 0.6-1.2% for PLP and 0.9-1.8% for 4-PA. Run-to-run (n=23) reproducibilities were 3.6-6.7% for PLP and 3.7-5.6% for 4-PA. Relative detection (3sigma(0)) and quantitation (10sigma(0)) limits were 0.3 and 0.9 nmol/L, respectively, for both PLP and 4-PA using a 10-microl sample injection volume. Analytical recoveries ranged from 97 to 102%. Patient-matched serum and plasma specimens (n=25) were analyzed to evaluate specimen-type bias. Of the plasma types evaluated, heparinized plasma introduced the lowest relative bias for PLP (-5.3%) and minimal bias for 4-PA (-2.3%) compared with serum. Ethylenediaminetetraacetic acid (EDTA) plasma showed the lowest bias for 4-PA (0.7%) but a relatively high bias for PLP (13.0%) due to a chromatographic interference. Human serum samples from a non-representative population subset (n=303) were commensurate with values published for other vitamin B(6) HPLC methods. These values gave geometric means of 42.4 nmol/L for PLP and 27.3 nmol/L for 4-PA. Medians for PLP and 4-PA were 40.1 and 21.8 nmol/L, respectively. The high sensitivity, precision, and throughput of this method, combined with its minimal serum specimen (150 microl) and sample injection

  17. Intracellular adenosine 3',5'-phosphate formation is essential for down-regulation of surface adenosine 3',5'-phosphate receptors in Dictyostelium

    Van Haastert, Peter J. M.

    1994-01-01

    Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP receptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phosphorylation, sequestration and down-regulation of the surface receptors. The role of intracellular cAMP in down-regulation of surface receptors was investigated. Down-regulation of receptors does not occur under conditio...

  18. Inhibitor design for ribonuclease A: the binding of two 5 '-phosphate uridine analogues

    Tsirkone, Vicky G.; Dossi, Kyriaki; Drakou, Christina; Zographos, Spyros E.; Kontou, Maria; Leonidas, Demetres D.

    2009-01-01

    The structure of ribonuclease A in complex with uridine 5′-phosphate and uridine 5′-diphosphate has been determined at 1.4 Å resolution in order to facilitate the rational design of selective and potent inhibitors.

  19. Hydrolysis of pyridoxal-5'-phosphate in plasma in conditions with raised alkaline phosphate.

    Anderson, B B; O'Brien, H.; Griffin, G E; Mollin, D. L.

    1980-01-01

    Hydrolysis of pyridoxal phosphate in plasma was demonstrated in patients with liver disease and other conditions with raised alkaline phosphatase, and this usually closely paralleled the alkaline phosphatase level, whether of liver or bone origin. The endogenous plasma pyridoxal phosphate was inversely related to the alkaline phosphatase, and plasma hydrolysis of pyridoxal phosphate may at least in part be responsible. Very large doses of vitamin B6 may be necessary to compensate for this hyd...

  20. Chemical modification of human muscle aldose reductase by pyridoxal 5'-phosphate

    Aldose reductase (ALR2) is a monomeric oxidoreductase (Mr, 37,000). This enzyme catalyzes the reduction of a wide variety of aliphatic and aromatic aldehydes to their corresponding alcohols. The ability to reduce D-glucose and utilize NADH distinguishes ALR2 from aldehyde reductase (ALR1) which is exclusively NADPH-dependent. As part of a study to determine active site residues critical for binding and catalysis they have investigated the behavior of ALR2 with pyridoxal phosphate (PLP). In contrast to ALR1, which is inactivated by PLP, the reaction of ALR2 with PLP results in a 2-3 fold activation with the incorporation of 1 mol of PLP/mol enzyme. However, despite a 3-fold increase in k/sub cat/, there is also a 13-14 fold increase in the Km for both coenzyme and substrate and catalytic efficiency (k/sub cat//Km) is actually decreased. Reaction of ALR2 with 3[H] PLP followed by digestion with endoproteinase Lys-C enabled the separation and purification by HPLC of a peptide containing a single pyridoxyllysine residue. The sequence of this 32 residue peptide is highly homologous with a peptide similarly obtained from pig and human ALR1 and is identical with one from pig ALR2. In all four enzymes, pig ALR1, ALR2; human ALR1, ALR2, a tetrapeptide containing the pyridoxylated lysine (I-P-K-S) shows absolute identity. Thus, despite differences in substrate and coenzyme specificity, the active site in both ALR1 and ALR2 is relatively conserved

  1. Ribose-5-Phosphate Biosynthesis in Methanocaldococcus jannaschii Occurs in the Absence of a Pentose-Phosphate Pathway

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

    2005-01-01

    Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns ...

  2. A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

    Mindy I Davis

    Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

  3. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

    Gelato, K.; Tauber, M; Ong, M; Winter, S.; Hamada, K; Sindlinger, J.; Lemak, A.; Bultsma, Y.; Houliston, S.; Schwarzer, D; Divecha, N.; C. Arrowsmith; Fischle, W

    2014-01-01

    UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occ...

  4. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

    Gelato, Kathy A; Tauber, Maria; Ong, Michelle S; Winter, Stefan; Hiragami-Hamada, Kyoko; Sindlinger, Julia; Lemak, Alexander; Bultsma, Yvette; Houliston, Scott; Schwarzer, Dirk; Divecha, Nullin; Arrowsmith, Cheryl H; Fischle, Wolfgang

    2014-06-19

    UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates. PMID:24813945

  5. D-fructose-6-phosphate aldolase in organic synthesis: cascade chemical-enzymatic preparation of sugar-related polyhydroxylated compounds.

    Concia, Alda Lisa; Lozano, Carles; Castillo, José A; Parella, Teodor; Joglar, Jesús; Clapés, Pere

    2009-01-01

    Novel aldol addition reactions of dihydroxyacetone (DHA) and hydroxyacetone (HA) to a variety of aldehydes catalyzed by D-fructose-6-phosphate aldolase (FSA) are presented. In a chemical-enzymatic cascade reaction approach, 1-deoxynojirimycin and 1-deoxymannojirimycin were synthesized starting from (R)- and (S)-3-(N-Cbz-amino)-2-hydroxypropanal, respectively. Furthermore, 1,4-dideoxy-1,4-imino-D-arabinitol and 1,4,5-trideoxy-1,4-imino-D-arabinitol were prepared from N-Cbz-glycinal. 1-Deoxy-D-xylulose was also synthesized by using HA as the donor and either 2-benzyloxyethanal or 2-hydroxyethanal as acceptors. In both cases the enzymatic aldol addition reaction was fully stereoselective, but with 2-hydroxyethanal 17 % of the epimeric product at C2, 1-deoxy-D-erythro-2-pentulose, was observed due to enolization/epimerization during the isolation steps. It was also observed that D-(-)-threose is a good acceptor substrate for FSA, opening new synthetic possibilities for the preparation of important novel complex carbohydrate-related compounds from aldoses. To illustrate this, 1-deoxy-D-ido-hept-2-ulose was obtained stereoselectively by the addition of HA to D-(-)-threose, catalyzed by FSA. It was found that the reaction performance depended strongly on the donor substrate, HA being the one that gave the best conversions to the aldol adduct. The examples presented in this work show the valuable synthetic potential of FSA for the construction of chiral complex polyhydroxylated sugar-type structures. PMID:19222084

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

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

    2010-06-01

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

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

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

    2016-07-01

    The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812. PMID:26765812

  8. Complexation of neptunium(V) by salicylate, phthalate and citrate ligands in a pH 7.5 phosphate buffered system

    Conditional stability constants, enthalpies and entropies of complexation at pH 7.5 and ionic strength 0.1 have been determined for neptunium(V) complexes of phosphate, salicylate, phthalate and citrate. Results are given and discussed. At pH 7.5 salicylate does not form a complex with neptunium(V) due to the low charge density of the NpO2+ ion and incomplete ionization of the salicylate ion. In all cases, only 1:1 complexes were identified. (U.K.)

  9. Characterization of the TDP-d-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-d-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate†

    Kharel, Madan K.; Lian, Hui; Rohr, Jürgen

    2011-01-01

    Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (d-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-d-ravidosamine biosynt...

  10. Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state 15N nuclear magnetic resonance

    Inhibition of alanine racemase from the Gram-positive bacterium Bacillus stearothermophilus by (1-aminoethyl)phosphonic acid (Ala-P) proceeds via a two-step reaction pathway in which reactivation occurs very slowly. In order to determine the mechanism of inhibition, the authors have recorded low-temperature, solid-state 15N NMR spectra from microcrystals of the [15N]Ala-P-enzyme complex, together with spectra of a series of model compounds that provide the requisite database for the interpretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at ∼ 150 ppm, which conclusively demonstrates the presence of a protonated Ala-P-PLP aldimine and thus clarifies the structure of the enzyme-inhibitor complex. They also report the pH dependence of Ala-P binding to alanine racemase

  11. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed a...

  12. Cloning and Analysis of cDNA Encoding Key Enzyme DXR in Diterpenoids Biosynthesis Pathway from Rabdosiae Rubescentis Herba%冬凌草1-脱氧木酮糖-5-磷酸还原异构酶(DXR)基因克隆与分析

    苏秀红; 尹磊; 陈随清

    2016-01-01

    目的:为研究冬凌草二萜类合成的相关基因,在冬凌草转录组信息数据的基础之上,以冬凌草无菌苗为研究材料,克隆冬凌草二萜类合成的关键酶1-脱氧木酮糖-5-磷酸还原异构酶(l-deoxy-D-lxyluloses-phosphatereduetoisomerase,DXR)基因.方法:采用逆转录PCR技术克隆冬凌草DXR基因,实时荧光定量PCR法分析其组织表达模式.结果:DXR cDNA基因全长1 500 bp,DXR基因开放阅读框为1 422 bp,编码473个氨基酸组成的蛋白质序列,理论相对分子质量为51.39 kDa,等电点为6.09,是一种亲水性蛋白.DXR在茎中表达量相对较高,在愈伤组织中表达量最低.结论:研究结果为深入研究冬凌草DXR酶的活性和功能及为冬凌草二萜类化合物的生物合成机制、优良基因挖掘奠定基础.%Objective:To study the genes related to the synthesis of diterpenoid.Based on the data of transcriptome sequencing,cDNAs encoding 1-deoxy-D-xylulose-5-phosphatereduetoisomerase (DXR) were obtained from the leaves of aseptic seedlings of Rabdosiae Rubescentis Herba.Method:DXR was obtained by reverse transcription PCR.Real-time quantitative PCR was used to detect the relative expression patterns of DXR in different tissues of Rabdosiae Rubescentis Herba.Result:Sequence analysis showed that the full-length cDNA of DXR was 1 500 bp and contains gene open reading frame (ORF) of 1 422 bp encoding 473 amino acids.The theoretical molecular weight was 51.39 kDa and the isoelectric point was predicted as 6.09,suggesting it was a type of hydrophilic protein.The expression pattern of the gene in different tissues was analyzed by Real-time fluorescence quantitative PCR.The results showed the expression of DXR was relatively high in the stem and the lowest in callus.Conclusion:The results will provide a basis for studying the activity and function of DXR from Rabdosiae Rubescentis Herba,and lay a foundation for biosynthesis and gene mining of terpenoids.

  13. Host cells and methods for production of isobutanol

    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.

  14. Protein (Cyanobacteria): 410899 [PGDBj - Ortholog DB

    Full Text Available LFAIPIASFWGRYSQIWAISHYPYLNKEGSSKFLHKKNWRGFLIESIPSYAFLSFLIFILINIDISIISTPNLIIGIIVGFLPALIIPHLLARRLGGHSGDSYGASVVLVETCMLIIFSIILPAS ... ...GVLQVSVLILLLQLQWPNESMPFIAIALGLWITGGIHVDGLMDTADGIAAGPSRCIEAMKDSRIGASGIIALTINLLLQIAALFKLRLLI...1 cobalamin-5-phosphate synthase Prochlorococcus marinus subsp. marinus str. CCMP1375 MSSPNWLKDLTGAWLFYTVFPKLTRINPRFERIARFSPLIGVLI

  15. Main: 1H1Y [RPSD[Archive

    Full Text Available 1H1Y イネ Rice Oryza sativa L. D-Ribulose-5-Phosphate ... 3-Epimerase Oryza Sativa Molecule: D-Ribul ... ose-5-Phosphate ... 3-Epimerase; Chain: A, B; Engineered: Yes Isomeras ... e 5.1.3.1 (D-Ribulose-5-Phosphate ... 3-Epimerase) S.Jelakovic, G.E.Schulz S.Jelakovic, ...

  16. Main: 1H1Z [RPSD[Archive

    Full Text Available 1H1Z イネ Rice Oryza sativa L. D-Ribulose-5-Phosphate ... 3-Epimerase Oryza Sativa Molecule: D-Ribul ... ose-5-Phosphate ... 3-Epimerase; Chain: A, B; Engineered: Yes Isomeras ... e 5.1.3.1 (D-Ribulose-5-Phosphate ... 3-Epimerase) S.Jelakovic, G.E.Schulz S.Jelakovic, ...

  17. pKNOT: the protein KNOT web server

    Lai, Yan-Long; Yen, Shih-Chung; Yu, Sung-Huan; Hwang, Jenn-Kang

    2007-01-01

    Knotted proteins are more commonly observed in recent years due to the enormously growing number of structures in the Protein Data Bank (PDB). Studies show that the knot regions contribute to both ligand binding and enzyme activity in proteins such as the chromophore-binding domain of phytochrome, ketol–acid reductoisomerase or SpoU methyltransferase. However, there are still many misidentified knots published in the literature due to the absence of a convenient web tool available to the gene...

  18. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  19. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

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

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

    2016-06-15

    The studies of 3-deoxy-d-manno-octulosonic acid (KDO) have been hindered due to its limited availability. Herein, an efficient enzymatic system for the facile synthesis of KDO from easy-to-get starting materials is described. In this one-pot three-enzyme (OPME) system, d-ribulose 5-phosphate, which was prepared from d-xylose, was employed as starting materials. The reaction process involves the isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by d-arabinose 5-phosphate isomerase (KdsD), the aldol condensation of d-arabinose 5-phosphate and phosphoenolpyruvate (PEP) catalyzed by KDO 8-phosphate synthetase (KdsA), and the hydrolysis of KDO-8-phosphate catalyzed by KDO 8-phosphate phosphatase (KdsC). By using this OPME system, 72% isolated yield was obtained. The obtained KDO was further transferred to lipid A by KDO transferase from Escherichia coli (WaaA). PMID:27173798

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

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Lange, Bernd Markus (Pullman, WA); McCaskill, David G. (Pullman, WA)

    2001-01-01

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

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

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

    2012-01-17

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

  3. Synthesis, Structure and Biological Activities of Some Novel N-(4,6-Disubstituted-pyrimidin-2-yl)-N'-(trifluoromethylphenyl)-guanidine Derivatives

    HE Feng-Qi; LIU Xing-Hai; WANG Bao-Lei; LI Yong-Hong; LI Zheng-Ming

    2008-01-01

    A novel series of guanidine derivatives were designed, synthesized and confirmed by FTIR, MS, 1H NMR and elemental analysis. The single crystal structure of 11b was determined by X-ray diffraction. Herbicidal activities of these guanidine derivatives were evaluated through barnyardgrass and rape cup tests, among which compounds lla, lib, and lie against Brassica campestris reached 71.2%, 86.7%, and 86.9% at 100 μg·mL-1 respectively. The preliminary ketol-acid reductoisomerase test showed that the synthesized compounds had weak activities.

  4. Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine.

    Pfefferkorn, E R; Bzik, D J; Honsinger, C P

    2001-12-01

    In contrast to the cytocidal effect of 6-thiopurines on mammalian cells, the action of 6-thioxanthine on Toxoplasma gondii was only parasitostatic. 6-Thioxanthine was a substrate of the parasite's hypoxanthine-guanine phosphoribosyltransferase. That enzyme converted 6-thioxanthine to 6-thioxanthosine 5'-phosphate which accumulated to near millimolar concentrations within parasites incubated intracellularly in medium containing the drug. 6-Thioxanthosine 5'-phosphate was the only detectable metabolite of 6-thioxanthine. The absence of 6-thioguanine nucleotides explains the lack of a parasitocidal effect because the incorporation of 6-thiodeoxyguanosine triphosphate into DNA is the mechanism of the lethal effect of 6-thiopurines on mammalian cells. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate incorporated more labeled hypoxanthine or xanthine into their nucleotide pools than did control parasites. The basis for this increased nucleobase salvage remains unexplained. It was not due to up-regulation of hypoxanthine-guanine phosphoribosyltransferase and could not be explained by reduced use of labeled nucleotides for nucleic acid synthesis. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate used labeled hypoxanthine almost entirely to make adenine nucleotides while control parasites made both adenine and guanine nucleotides. Both extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate and control parasites efficiently used labeled xanthine to make guanine nucleotides. These observations suggested that inosine 5'-phosphate-dehydrogenase was inhibited while guanosine 5'-phosphate synthase was not. Assay of inosine 5'-phosphate dehydrogenase in soluble extracts of T. gondii confirmed that 6-thioxanthosine 5'-phosphate was an inhibitor. We conclude that 6-thioxanthine blocks the growth of T. gondii by a depletion a guanine

  5. Pyridoxine (Vitamin B6)

    ... used in combination with other B vitamins in vitamin B complex products. You may remember a prescription medication called ... Adermine Chlorhydrate, Adermine Hydrochloride, B Complex Vitamin, B6, Chlorhydrate ... Pyridoxal, Pyridoxal Phosphate, Pyridoxal 5 Phosphate, Pyridoxal-5- ...

  6. Protein: MPA5 [TP Atlas

    Full Text Available MPA5 Pyrimidine biosynthesis Tc00.1047053507059.60 Orotidine-5-phosphate decarboxylase/orotate p ... phoribosyltransferase, putative 353153 Trypanosoma cruzi ... (strain CL Brener) 3542677 Q4DBC5 ...

  7. ClEST cluster :Cl_singleton1944 [ClEST

    Full Text Available Cl_singleton1944 deoxyribose-phosphate ... aldolase Cimex lectularius nuclear gene 535 GATATCTGAAATG ... TG mb07072 1 PREDICTED: similar to 2-deoxyribose-5-phosphate ... aldolase homolog [Tribolium castaneum] XP_972358 7 ...

  8. Futile xylitol cycle in Lactobacillus casei.

    Hausman, S Z; Thompson, J; London, J

    1984-01-01

    A futile xylitol cycle appears to be responsible for xylitol-mediated inhibition of growth of Lactobacillus casei Cl-16 at the expense of ribitol. The gratuitously induced xylitol-specific phosphoenolpyruvate-dependent phosphotransferase accumulates the pentitol as xylitol-5-phosphate, a phosphatase cleaves the latter, and an export system expels the xylitol. Operation of the cycle rapidly dissipates the ribitol-5-phosphate pool (and ultimately the energy supply of the cell), thereby producin...

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

    Johan Puts

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

  10. Design,Synthesis and Biological Activity of Ethyl 2-(N-Substituted-arylsulfonamido)-2-oxoacetate

    WANG Bao-lei; WU Jing; HE Feng-qi; LI Yon-ghong; LI Zheng-ming

    2008-01-01

    Thirteen new ethyl 2-(N-substituted-arylsulfonamido)-2-oxoacetates(3a-3m),based on the structure of Ketol-acid reductoisomerase(KARI) inhibitor IpOHA,were designed and synthesized,Their structures were established on the basis of 1H NMR,IR,MS,and elemental analyses,The bioassay result reveals that the structural changes from hydroxyl group on the N atom of IpOHA to arylsulfonyl groups does not enhance the inhibitory activity of the compounds to KARI in vitro,Compounds 3c,3h,3k,and 3m are more effective than IpOHA against the monocotyledonous barnygrass at 100 μg/mL in herbicidal tests.

  11. Synthesis of mixtures of [2H6]myo-inositol monophosphates suitable as internal standards for quantitative GC/MS and a simple synthesis of DL-myo-inositol-1-phosphate

    A mixture of DL-[2H6]myo-inositol-1- and -4-phosphate and (meso) myo-inositol-2-and -5-phosphate is readily prepared on a 100 milligram scale from the mixed isomers of di-O-cyclohexylidene-[2H6]myo-inositol by phosphorylation with tetrabenzylpyrophosphate. The deblocked [2H6]myl-inositol monophosphate isomers are obtained where the relative amounts of racemic myo-inositol-1-phosphate > -4-phosphate > -2-phosphate > -5-phosphate. In addition a simple synthesis of DL-myo-inositol-1-phosphate was developed which was found to be unsatisfactory for the preparation of deuterium-labeled product. (Author)

  12. Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

    Bisp, Marianne R; Bor, Mustafa Vakur; Heinsvig, Else-Marie;

    2002-01-01

    vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers...... were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate...

  13. Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp paracasei CHCC 2115

    Thage, B.V.; Rattray, F.P.; Laustsen, M.W.;

    2004-01-01

    temperature optima of 7.3 and 43degreesC, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl). Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of...

  14. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

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

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-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...

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

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

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

    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.

  17. SwissProt search result: AK106698 [KOME

    Full Text Available AK106698 002-114-D11 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 9e-20 ...

  18. SwissProt search result: AK061902 [KOME

    Full Text Available AK061902 001-041-H06 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-14 ...

  19. SwissProt search result: AK119895 [KOME

    Full Text Available AK119895 002-180-C05 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 5e-15 ...

  20. SwissProt search result: AK064089 [KOME

    Full Text Available AK064089 002-100-D02 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 8e-25 ...

  1. SwissProt search result: AK107554 [KOME

    Full Text Available AK107554 002-130-B02 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 2e-18 ...

  2. SwissProt search result: AK103526 [KOME

    Full Text Available AK103526 J033131L11 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 4e-23 ...

  3. SwissProt search result: AK061521 [KOME

    Full Text Available AK061521 006-310-B05 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 6e-19 ...

  4. SwissProt search result: AK064221 [KOME

    Full Text Available AK064221 002-104-G01 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-16 ...

  5. SwissProt search result: AK067006 [KOME

    Full Text Available AK067006 J013093O09 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-17 ...

  6. SwissProt search result: AK105961 [KOME

    Full Text Available AK105961 001-205-E02 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-23 ...

  7. SwissProt search result: AK069342 [KOME

    Full Text Available AK069342 J023013K12 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 4e-14 ...

  8. SwissProt search result: AK103774 [KOME

    Full Text Available AK103774 J033143M23 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 1e-19 ...

  9. SwissProt search result: AK101194 [KOME

    Full Text Available AK101194 J033030J11 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-16 ...

  10. SwissProt search result: AK064342 [KOME

    Full Text Available AK064342 002-107-H07 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-18 ...

  11. SwissProt search result: AK109784 [KOME

    Full Text Available AK109784 002-147-C09 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 3e-17 ...

  12. SwissProt search result: AK106518 [KOME

    Full Text Available AK106518 002-107-C02 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 6e-20 ...

  13. SwissProt search result: AK109450 [KOME

    Full Text Available AK109450 001-114-F08 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 1e-24 ...

  14. SwissProt search result: AK108373 [KOME

    Full Text Available AK108373 002-142-D10 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 4e-20 ...

  15. SwissProt search result: AK059748 [KOME

    Full Text Available AK059748 006-203-A07 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 5e-19 ...

  16. SwissProt search result: AK102879 [KOME

    Full Text Available AK102879 J033112G11 (Q8ZCX5) Phosphate import ATP-binding protein pstB 1 (EC 3.6.3.27) (Phosphate...-transporting ATPase 1) (ABC phosphate transporter 1) (Phosphate-specific transport component 1) PSTB1_YERPE 1e-18 ...

  17. Consequences of a Deficit in Vitamin B-6 Biosynthesis de Novo for Hormone Homeostasis and Root Development in Arabidopsis

    Boycheva, S.; Dominguez, A.; Rolčík, Jakub; Dominguez, T.; Fitzpatrick, T.B.

    2015-01-01

    Roč. 167, č. 1 (2015), s. 102-117. ISSN 0032-0889 Institutional support: RVO:61389030 Keywords : PYRIDOXAL 5'-PHOSPHATE SYNTHASE * EARLY SEEDLING DEVELOPMENT * AUXIN RESPONSE ELEMENTS Subject RIV: CE - Biochemistry Impact factor: 6.841, year: 2014

  18. Probe data: 6356 [RED

    Full Text Available R01-072960-1AR RED C28243 C97586 C60441 CH0441 >(Q42942) URIDINE 5'-MONOPHOSPHATE SYNTHASE (UMP ... SYNTHASE) (OROTATE PHOSPHORIBOSYLTRANSFERASE (EC ... 2.4.2.10) AND OROTIDINE 5'-PHOSPHATE DEC ARBOXYLASE ... (EC ... 4.1.1.23)) (FRAGMENT). &NTU22260_1(U22260|pid:g747 ...

  19. EST data: 1908 [RED

    Full Text Available CH0441 C28243 C97586 C60441 1 D DPlate 020 D11 5' GCG AGT AAT CCA GCC GAG AC 3' 5' CAA ACC CAA T ... (UMP SYNTHASE) (OROTATE PHOSPHORIBOSYLTRANSFERASE (EC ... 2.4.2.10) AND OROTIDINE 5'-PHOSPHATE DEC ARBOXYLASE ... (EC ... 4.1.1.23)) (FRAGMENT). &NTU22260_1(U22260|pid:g747 ...

  20. Nuclear PI5P, Uhrf1, and the Road Not Taken

    Reynoird, Nicolas; Gozani, Or

    2014-01-01

    In this issue of Molecular Cell, Gelato et al (2014) identify the signaling molecule phosphatidylinositol-5-phosphate (PI5P) as an allosteric regulator that determines the mode of chromatin binding for the DNA methylation maintenance factor Uhrf1. This work links nuclear lipids to chromatin signaling in the maintenance of DNA methylation and epigenetic regulation.

  1. Nuclear PI5P, Uhrf1, and the road not taken.

    Reynoird, Nicolas; Gozani, Or

    2014-06-19

    In this issue of Molecular Cell, Gelato et al. (2014) identify the signaling molecule phosphatidylinositol 5-phosphate (PI5P) as an allosteric regulator that determines the mode of chromatin binding for the DNA methylation maintenance factor Uhrf1. This work links nuclear lipids to chromatin signaling in the maintenance of DNA methylation and epigenetic regulation. PMID:24950375

  2. Polyol synthesis in Aspergillus niger : influence of oxygen availability, carbon and nitrogen sources on the metabolism

    Diano, Audrey; Bekker-Jensen, S; Dynesen, Jens Østergaard;

    2006-01-01

    , erythritol, xylitol, and arabitol were produced as carbon storage compounds when the flux through the PP pathway exceeded the need in ribulose-5-phosphate for the biomass synthesis. Glycerol, erythritol, and xylitol seem to be involved in osmoregulation. Mannitol was produced when the catabolic reduction of...

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

    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...

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

    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

  5. The design, synthesis of amide KARI inhibitors and their biological activities

    Baolei WANG; Yi MA; Yonghong LI; Suhua WANG; Zhengming LI

    2009-01-01

    Ketol-acid reductoisomerase(KARI) is a promising target for the design of herbicides yet there are only few reports on the molecular design of KARI inhibitors. In this paper, based on the reported 0.165 nm high resolution crystal structure of the spinach KARI complex, 279 molecules with low binding energy toward KARI were obtained from an MDL/ACD 3D database search using the program DOCK 4.0. According to the structural information of 279 molecules provided, some amide compounds have been designed and synthesized. The bioassay results show that most of these amides had inhibitory activity to rice KARI at a test concentration of 200 μg/mL. Among which eight amides, compounds 1 and 6 show 57.4% and 48.1% inhibitory activity to KARI. The herbicidal activities of these amides were further investigated on di-cotyledonous rape (Brassica campestris) and mono-cotyledonous bar-nyardgrass (Echinochloa crusgalli). Compounds 1 and 6 were more favorable than others and showed 52.0% and 72.6% inhibitory activity on rape root at 100 μg/mL concentration, respectively. These amides could be further optimized for finding more potent candidates.

  6. α,β-Dicarbonyl reduction is mediated by the Saccharomyces Old Yellow Enzyme.

    van Bergen, Barry; Cyr, Normand; Strasser, Rona; Blanchette, Maxime; Sheppard, John D; Jardim, Armando

    2016-08-01

    The undesirable flavor compounds diacetyl and 2,3-pentanedione are vicinal diketones (VDKs) formed by extracellular oxidative decarboxylation of intermediate metabolites of the isoleucine, leucine and valine (ILV) biosynthetic pathway. These VDKs are taken up by Saccharomyces and enzymatically converted to acetoin and 3-hydroxy-2-pentanone, respectively. Purification of a highly enriched diacetyl reductase fraction from Saccharomyces cerevisiae in conjunction with mass spectrometry identified Old Yellow Enzyme (Oye) as an enzyme capable of catalyzing VDK reduction. Kinetic analysis of recombinant Oye1p, Oye2p and Oye3p isoforms confirmed that all three isoforms reduced diacetyl and 2,3-pentanedione in an NADPH-dependent reaction. Transcriptomic analysis of S. cerevisiae (ale) and S. pastorianus (lager) yeast during industrial fermentations showed that the transcripts for OYE1, OYE2, arabinose dehydrogenase (ARA1), α-acetolactate synthase (ILV2) and α-acetohydroxyacid reductoisomerase (ILV5) were differentially regulated in a manner that correlated with changes in extracellular levels of VDKs. These studies provide insights into the mechanism for reducing VDKs and decreasing maturation times of beer which are of commercial importance. PMID:27400981

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

    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.

  8. Synthesis and characterization of chitosan phosphopyridoxal Schiff base derivative in ionic liquid%离子液体中壳聚糖磷酸吡哆醛席夫碱衍生物的合成与表征

    李克让; 徐民; 张帅; 刘蒲

    2013-01-01

    5-Phosphate pyridoxal (PPL) is the active coenzyme form of vitamin B6, acting as a coenzyme in a multitude of biochemical processes, therefore chitosan derivatives containing pyridoxal phosphate skeleton will provide an important material for the application of chitosan in the emerging biological function material. Chitosan phosphopyridoxal Schiff base derivative was synthesized by the condensation reaction of chitosan and 5-phosphate pyridoxal in an ionic liquid, 1-butyl-3-methylimidazolium chloride (BmimCl). The product was characterized by Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), XRD and TGA. The results showed that phosphopyridoxal group was successfully introduced onto the chitosan chain. It was found that degree of substitution of modified chitosan could reach 16.3%.

  9. [The vitamin B6 allowance of hypertension patients and the effect of dietotherapy].

    Duleeva, V L; Piatnitskaia, I N; Bogdanov, N G

    1990-01-01

    A total of 111 patients with essential hypertension (the II stage) and obesity (the II degree) were investigated for providing with vitamin B6. The functional methods used for the vitamin assay (ACT activity of red blood cells and pyrodoxale-5-phosphate effect) have revealed significant vitamin B6 deficiency in 81.1% of the patients. Vitamin B6 deficiency was intensified in the course of the dietotherapy. Correction of vitamin B6 deficiency with a therapeutic dose of pyridoxine (20 mg/day) during 20-22 days, in the presence of the diet, has promoted optimization of providing with vitamin B6: normalization of pyrodoxale-5-phosphate effect. The hypotensive effect and decrease of excessive body mass in patients who received dietotherapy and pyridoxine (20 mg/day) were more pronounced than in those who received the same diet and the multivitamin "Undevitum". PMID:2399675

  10. Molecular Docking and NMR Binding Studies to Identify Novel Inhibitors of Human Phosphomevalonate Kinase

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

    2012-01-01

    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 1H-15N Heteronuclear Single Quantum Coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain d...

  11. Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

    Oikonomakos, Nikos G.; Zographos, Spyros E.; Tsitsanou, K. E.; Johnson, L N; Acharya, K. R.

    1996-01-01

    It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of nativ...

  12. Controlling reaction specificity in pyridoxal phosphate enzymes

    Michael D Toney

    2011-01-01

    Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carb...

  13. Malonate-based inhibitors of mammalian serine racemase: Kinetic characterization and structure-based computational study

    Vorlová, Barbora; Nachtigallová, Dana; Jirásková-Vaníčková, Jana; Ajani, Haresh; Jansa, Petr; Řezáč, Jan; Fanfrlík, Jindřich; Otyepka, M.; Hobza, Pavel; Konvalinka, Jan; Lepšík, Martin

    2015-01-01

    Roč. 89, Jan 7 (2015), s. 189-197. ISSN 0223-5234 R&D Projects: GA ČR GBP208/12/G016 Grant ostatní: GA MŠk(CZ) ED2.1.00/03.0058 Institutional support: RVO:61388963 Keywords : NMDA receptor * pyridoxal-5 '-phosphate-dependent enzyme * human/mouse serine racemase * malonate-based inhibitors * semiempirical quantum mechanical calculations Subject RIV: CE - Biochemistry Impact factor: 3.447, year: 2014

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

    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. PMID:12182339

  15. Evaluation of vitamin B6 intake and status of 20- to 64-year-old Koreans

    Kim, Young-Nam; Cho, Youn-OK

    2014-01-01

    BACKGROUND/OBJECTIVES Recent research regarding vitamin B6 status including biochemical index is limited. Thus, this study estimated intakes and major food sources of vitamin B6; determined plasma pyridoxal 5'-phosphate (PLP); and assessed vitamin B6 status of Korean adults. MATERIALS/METHODS Three consecutive 24-h diet recalls and fasting blood samples were collected from healthy 20- to 64-year-old adults (n = 254) living in the Seoul metropolitan area, cities of Kwangju and Gumi, Korea. Vit...

  16. Transaldolase Deficiency: Liver Cirrhosis Associated with a New Inborn Error in the Pentose Phosphate Pathway

    Verhoeven, Nanda M; Huck, Jojanneke H. J.; Roos, Birthe; Struys, Eduard A.; Salomons, Gajja S; Douwes, Adriaan C.; van der Knaap, Marjo S.; Jakobs, Cornelis

    2001-01-01

    This article describes the first patient with a deficiency of transaldolase (TALDO1 [E.C.2.2.1.2]). Clinically, the patient presented with liver cirrhosis and hepatosplenomegaly during early infancy. In urine and plasma, elevated concentrations of ribitol, d-arabitol, and erythritol were found. By incubating the patient's lymphoblasts and erythrocytes with ribose-5-phosphate and subsequently analyzing phosphate sugar metabolites, we discovered a deficiency of transaldolase. Sequence analysis ...

  17. Assembly of the eukaryotic PLP-synthase complex from plasmodium and activation of the Pdx1 enzyme

    Guedez, Gabriela; Hipp, Katharina; Windeisen, Volker; Derrer, Bianca; Gengenbacher, Martin; Boettcher, Bettina; Sinning, Irmgard; Kappes, Barbara; Tews, Ivo

    2012-01-01

    Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B6 (pyridoxal 5-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alter...

  18. GenBank blastx search result: AK060768 [KOME

    Full Text Available AK060768 001-033-A12 AF160811.1 Bacillus stearothermophilus L-arabinose transport, ATP binding protein (ara...G), L-arabinose membrane permease (araH), AraR (araR), L-ribulose 5-phosphate 4-epimerase (ara...D), L-ribulokinase (araB), L-arabinose isomerase (araA), and IS5377 transposase genes, complete cds.|BCT BCT 9e-14 +3 ...

  19. Xylitol-mediated transient inhibition of ribitol utilization by Lactobacillus casei.

    London, J; Hausman, S

    1982-01-01

    The growth of Lactobacillus casei strain Cl-16 at the expense or ribitol was inhibited if the non-metabolizable substrate xylitol was included in the medium at concentrations of 6 mM or greater. At these concentrations, xylitol, did not competitively inhibit ribitol transport. The cessation of growth was caused by the intracellular accumulation of xylitol-5-phosphate, which occurred because growth on ribitol had gratuitously induced a functional xylitol-specific phosphotransferase system but ...

  20. Effects of xylitol on metabolic parameters and visceral fat accumulation

    Amo, Kikuko; Arai, Hidekazu; Uebanso, Takashi; Fukaya, Makiko; Koganei, Megumi; Sasaki, Hajime; Yamamoto, Hironori; Taketani, Yutaka; Takeda, Eiji

    2011-01-01

    Xylitol is widely used as a sweetener in foods and medications. Xylitol ingestion causes a small blood glucose rise, and it is commonly used as an alternative to high-energy supplements in diabetics. In previous studies, a xylitol metabolite, xylulose-5-phosphate, was shown to activate carbohydrate response element binding protein, and to promote lipogenic enzyme gene transcription in vitro; however, the effects of xylitol in vivo are not understood. Here we investigated the effects of dietar...

  1. Aldolases for Enzymatic Carboligation : Directed Evolution and Enzyme Structure-Function Relationship Studies

    Ma, Huan

    2015-01-01

    The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attenti...

  2. Intermediate und Enzyme des alternativen Terpenbiosyntheseweges

    Hecht, Stefan

    2007-01-01

    In der vorliegenden Arbeit wurden bisher unbekannte Reaktionsschritte der Biosynthese der universellen Terpenvorstufen Isopentenyldiphosphat und Dimethylallyldiphosphat über 1-Desoxy-D-xylulose-5-phosphat aufgeklärt. Hierzu wurde eine flexible enzymatische Synthese von Intermediaten des alternativen Terpenbiosynthesewegs entwickelt, die eine Markierung mit stabilen und radioaktiven Isotopen erlaubte. Mit Hilfe dieser Substrate wurde die katalytische Aktivität der beteiligten IspE und IspF Pro...

  3. Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

    Abbas, Charles A; Andriy A Sibirny

    2011-01-01

    Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pterid...

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

    The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30 a shows a carbon isotope effect k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 370C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine

  5. Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.

    Futaki, S; Ueno, H; Martinez del Pozo, A; Pospischil, M A; Manning, J M; Ringe, D; Stoddard, B; Tanizawa, K; Yoshimura, T; Soda, K

    1990-12-25

    In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild

  6. Overexpression of yeast ArDH gene in chloroplasts confers salinity tolerance in plants (abstract)

    Water stress due to salinity and drought is the main limiting factor for plant growth, productivity and quality. A common response to water deficit is the accumulation of osmoprotectants such as sugars and amino acids. In yeast, arabitol dehydrogenase is found responsible for the production of arabitol from ribulose-5-phosphate. All plants synthesize ribulose-5-phosphate via pentose pathway in chloroplasts.. Therefore, osmotolerance of the plants could be enhanced through metabolic engineering of chloroplasts by introducing ArDH gene into the plastome, which is responsible for the conversion of ribulose-5- phosphate to arabitol. Here we report high-level expression of arabitol dehydrogenase (ArDH) in chloroplasts. Homoplasmic transgenic plants were recovered on spectinomycin-containing regeneration medium. Transformed tobacco plants survived whereas non-transformed were severely stressed or killed when two weeks old seedlings were exposed to NaCl (up to 400 mM), suggesting a role for arabitol in salt tolerance. Seedlings survived up to five weeks on medium containing high salt concentrations (350-400 mM). Nevertheless, seedlings remained green and grew normal on concentrations up to 350 mM NaCl for several weeks. Hypothesis that membranes are protected under stress conditions due to the arabitol accumulation in chloroplasts, seedlings were grown in liquid medium containing polyethylene glycol (PEG, up to 6%). Seedlings were tolerant to 6% PEG, suggesting that ArDH enzyme protects membranes integrity under stress. Therefore, it is concluded that ArDH gene could be expressed in crop plants to withstand abiotic stresses. (author)

  7. 6-Phosphogluconate Dehydrogenase Mechanism: EVIDENCE FOR ALLOSTERIC MODULATION BY SUBSTRATE

    Hanau, Stefania; Montin, Katy; Cervellati, Carlo; Magnani, Morena; Dallocchio, Franco

    2010-01-01

    The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG D(V/K) and D(V) are 1.6...

  8. Peroxide-dependent amino acid oxidation and chemiluminescence catalysed by magnesium-pyridoxal phosphate-glutamate complex.

    Meyer, B U; Schneider, W; Elstner, E F

    1992-08-01

    Magnesium-pyridoxal-5'-phosphate-glutamate (MPPG) has been shown to ameliorate atherosclerotic symptoms in rabbits. In vitro, MPPG in the presence of peroxides such as cholesterolhydroperoxide or cumene hydroperoxide and Mn2+ ions produces "excited states" measurable as chemiluminescence or ethylene release from 1-aminocyclopropane-1-carboxylic acid (ACC). The reactions are stimulated synergistically by unsaturated fatty acids. Pyridoxal phosphate exhibits similar properties, but can be differentiated from the activities of MPPG or the sum of the components present in MPPG. PMID:1510700

  9. Development and validation of a HPLC method for the determination of metronidazole, oxytetracycline and furazolidone in veterinary formulations

    Violeta Tauber (Giugiu),; Viorica Chiurciu

    2014-01-01

    An isocratic reversed phase high performance liquid chromatographic method with DAD detection was developed for the analysis of metronidazole, oxytetracycline and furazolidone. The mobile phase consisted of pH 2.5 phosphate buffer solution, methanol and acetonitrile (70:18:12). The UV detection was carried out at 264 nm, and the flow rate was 1.100 mL/min. The separation was carried out on a Nucleosil C18 column, 5 µm 250 mmx4.6 mm, which was maintained at 25 0C. This method was validated by ...

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

    Graham, David E [ORNL

    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.

  11. Phytochrome control of gene expression in radish seedlings. I. Far-red light mediated stimulation of polyribosome formation and appearance of translatable mRNAs

    Illumination of radish seedlings with far-red light brings about an increase of the polyribosome content of cotyledons and hypocotyls. Poly(A)-containing RNA from cotyledons of both etiolated and far-red light-treated radish seedlings were isolated and translated in a rabbit reticulocyte lysate. The comparison of translation products showed that the relative levels of translatable mRNA coding for six abundant polypeptides were higher in the extracts of irradiated seedlings. The comparison of mRNA populations was also investigated with respect to organ specificity, polyribosomal localization and inhibition with the cap analoge, 7-methylguanosine 5'-phosphate. (author)

  12. Influence of vitamin B6-deficiency on the uracilnucleotid-metabolism in different organs of the rat and on the glycosylation of plasmamembrane-proteins

    Rieger, Kathrin

    2010-01-01

    Pyridoxal-5?-phosphate (PLP), the physiologically active form of vitamin B6, functions as a cofactor for enzymes in a variety of metabolic reactions, particulary the amino acid metabolism. In addition, a number of studies have demonstrated a new role of vitamin B6 as a modulator of gene expression. Vitamin B6-deficiency hereby resulted in an enhanced expression of a number of genes. Herein, the influence of vitamin B6-deficiency a) on the concentration of nucleotides and UDP-sugars in rat liv...

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

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

    2016-01-15

    Chemosensor L derived from vitamin B6 cofactor pyridoxal-5-phosphate was investigated for the selective detection of Cu(2+) and Fe(3+) in aqueous medium. Sensor L formed a 1:1 complex with Cu(2+) 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 Fe(3+) without any interference from other metal ions including Cu(2+). PMID:26344485

  14. Purification and characterization of Clostridium sticklandii D-selenocystine alpha, beta-lyase.

    Esaki, N; Seraneeprakarn, V; Tanaka, H.; Soda, K

    1988-01-01

    We have found a novel enzyme that decomposes D-selenocystine into pyruvate, ammonia, and elemental selenium in extracts of Clostridium sticklandii and C. sporogenes. The enzyme of C. sticklandii has been purified to homogeneity. It has a molecular weight of 74,000 and consists of two subunits identical in molecular weight (35,000). Pyridoxal 5'-phosphate is required as a cofactor. In addition to D-selenocystine, D-cystine, D-lanthionine, meso-lanthionine, and D-cysteine serve as substrates. H...

  15. Prediction of potential antimalarial targets of artemisinin based on protein information from whole genome of Plasmodium falciparum

    HAN LiPing; HUANG Qiang; NAN Peng; ZHONG Yang

    2009-01-01

    On the basis of the genomic data and protein pathway information about Plasmodium falciparum clone 3D7 from the NCBI taxonomy database and the KEGG database,eight key protein enzymes in the signal pathways were selected to perform molecular docking with artemisinin.The binding modes obtained from the molecular docking suggested that purine nucleoside phosphorylase (pfPNP),peptide deformylase (pfPDF),and ribose 5-phosphate isomerase (pfRpiA) may be involved in the antimalarial mode of action of artemisinin.Artemisinin exhibited its antimalarial activity probably by interfering with the metabolic pathways of purine,pyrimidine,methionine,glyoxylate and dicarboxylate,or pentose phosphate.

  16. Identification and Functional Analysis of the Gene Cluster for l-Arabinose Utilization in Corynebacterium glutamicum▿ †

    Kawaguchi, Hideo; Sasaki, Miho; Vertès, Alain A.; Inui, Masayuki; Yukawa, Hideaki

    2009-01-01

    Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (L...

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

    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 b...

  18. Single zymomonas mobilis strain for xylose and arabinose fermentation

    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.

  19. Pentose fermentation by recombinant zymomonas

    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.

  20. Recombinant Zymomonas for pentose fermentation

    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.

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

    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 +.

  2. Supply with the vitamins B1, B2 and B6 in carcinomas before and after radiotherapy

    In 108 breast cancer, 63 cervix carcinoma, 35 corpus carcinoma and 15 ovarial cancer patients the erythrocyte transketolase, gluthathione reductase and aspartate aminotransferase activity were determined as parameters for the supply with vitamin B1, B2 and B6 before and after radiotherapy. The effects of thiamine pyrophosphate determined in cancer patients were normal but the effects of flavin adenine dinucleotide and pyridoxal-5-phosphate were significantly increased compared to the controls. These results revealed radiation-induced disorders in the B2 metabolism and tumor-induced disorders in the B6 metabolism. Both disorders can be avoided by treatment with vitamin B complex. (author)

  3. Characterization of the different spectral forms of glutamate 1-semialdehyde aminotransferase by mass spectrometry

    Brody, S; Andersen, Jens S.; Kannangara, C G;

    1995-01-01

    Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulinate for the synthesis of chlorophyll, heme, and other tetrapyrrole pigments. The native enzyme from Synechococcus is pale yellow and has absorption maxima at 338 and 418 nm from vitamin B6. Yellow, colorless, and pink forms of the...... cofactor dissociated from the protein and gave a molecular weight of 46,401-46,418. Acetylenic GABA and NaBH4 were used for protein modification, and they reacted with the native and yellow enzymes but had no effect on the colorless enzyme. Pyridoxal 5'-phosphate bound covalently to the protein after NaBH4...

  4. DNA deoxyribophosphodiesterase.

    Franklin, W A; Lindahl, T

    1988-01-01

    A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deox...

  5. Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

    Gupta R, Baldock S J, Fielden P R, Prest J E, Grieve B D

    2011-01-01

    Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6 ± 0.1 min (n...

  6. 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.

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

    2016-03-01

    The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3',4,4',5,5')P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3',4,4',5,5')P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B-BiPh(3,3',4,4',5,5')P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed "moving metal" mechanism. PMID:26854536

  7. Method of preparing uridine-diphospho-14C-D-glucose of high molar activity and radiochemical purity by enzyme synthesis

    A new method is described of enzyme synthesis of uridine-diphospho-14C-D-glucose of high molar activity. After conversion of the initial 14C-D-glucose, the accompanying sugar phosphates and nucleoside-5'-phosphates are selectively hydrolyzed by the action of alkaline phosphatase in the presence of phenolphthalein as an indicator. Uridine-diphospho-14C-D-glucose is separated by paper chromatography from the reaction mixture thus modified. Synthesis yields range within 75% and 85% relative to the starting 14C-D-glucose. (B.S.)

  8. Enzymatic synthesis of radioisotope-labeled nucleotides from the corresponding labeled nitrogen bases

    The authors have developed a procedure that permits the production of nucleotides multiply labeled with various radioisotopes, with a molar radioactivity equal to the molar radioactivity of the original nitrogen bases. The methods of isolation and purification of the enzyme preparations were studied on the enzyme systems converting nitrogen bases to nucleoside triphosphates in the presence of phosphoribosyl pyrophosphate or ribose-5-phosphate. The presence of nucleotide impurities in the enzyme preparation was determined spectrophotometrically. The authors were able to practically avoid nucleotide impurities by salting out the protein fraction of interest with ammonium sulfate. The authors succeeded in reducing the content of nucleotides in the enzyme preparation by a factor of 40

  9. Via das pentoses fosfato na saúde e na doença: da disfunção metabólica aos biomarcadores

    Ramos, Rúben José Jesus Faustino

    2013-01-01

    Tese de mestrado, Análises Clínicas, Universidade de Lisboa, Faculdade de Farmácia, 2013 The Pentose Phosphate Pathway (PPP) fulfils two unique functions: (i) the formation of ribose-5-phosphate (R5P) for the synthesis of nucleotides, RNA and DNA, supporting cell growth and proliferation; and (ii) the formation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH carries chemical energy in the form of reducing power, being essential to the cellular oxidative de...

  10. Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

    Yan LIU; Hong WANG; He-Chun YE; Guo-Feng LI

    2005-01-01

    Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.

  11. The electrochemical behavior of N-n-undecyl-N'-(sodium-p- amino-benzenesulfonate) thiourea and its interaction with bovine serum albumin

    Hong Xia Luo; Yang Du; Zhi Xin Guo

    2008-01-01

    In pH 5.5 phosphate buffer solution, N-n-undecyl-N'-(sodium-p-amino-benzenesulfonate) thiourea (UPT) produced a pair ofredox peaks on the bare glassy carbon electrode. At the multi-walled carbon nanotube (MWNT) modified electrode, theelectrochemical behavior of UPT enhanced greatly. In the presence of bovine serum albumin (BSA), the peak currents ofUPT decreased linearly due to the formation of a super-molecular complex. This method was successfully applied to thedetermination of BSA in a bovine serum sample.2008 Hong Xia Luo. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  12. General Properties, Occurrence, and Preparation of Carbohydrates

    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.

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

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

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

    2016-05-15

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

  15. Inhibition of human pyridoxal kinase by 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI).

    Elsinghorst, Paul W; di Salvo, Martino L; Parroni, Alessia; Contestabile, Roberto

    2015-04-01

    2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is observed as a minor contaminant in caramel food colourings (E 150c). Feeding experiments with rodents have revealed a significant lymphopenic effect that has been linked to the presence of THI in these food colourings. Pyridoxal kinase inhibition by THI has been suggested, but not demonstrated, as a mode of action as it leads to lowered levels of pyridoxal-5'-phosphate, which are known to cause lymphopenia. Recently, THI was also shown to inhibit sphingosine-1-phosphate lyase causing comparable immunosuppressive effects and derivatives of THI are being developed for the treatment of rheumatoid arthritis in humans. Interestingly, sphingosine-1-phosphate lyase activity depends on pyridoxal-5'-phosphate, which in turn is provided by pyridoxal kinase. This report shows that THI does inhibit pyridoxal kinase with competitive and mixed-type non-competitive behaviour towards its two substrates, pyridoxal and ATP, respectively. The corresponding inhibition constants are in the low millimolar range. PMID:24899377

  16. Liquid chromatographic determination with fluorescence detection of B6 vitamers and riboflavin in milk and pharmaceuticals

    A simple, reliable and selective high performance liquid chromatographic method with fluorescence detection at different programmed wavelengths has been developed for the simultaneous analysis of B6 vitamers (pyridoxal 5'-phosphate, 4-pyridoxic acid, pyridoxal, pyridoxine and pyridoxamine) and Vitamin B2 in commercial vitaminized milk and in woman milk. The chromatographic separations were performed on a reversed phase octyl column by using a mobile phase consisting of sodium pentanesulfonate in 1% acetic acid-methanol-tetrahydrofuran under gradient elution conditions. The fluorescence intensity of pyridoxal 5'-phosphate was enhanced by post-column photochemical conversion, giving significantly different fluorescence spectra by a on-line photoreactor switched OFF and ON under irradiation at 254 nm. In addition, a simple and rapid method in isocratic conditions without the need of photochemical conversion was proposed for the analysis of Vitamin B6 and Vitamin B2 in pharmaceuticals. Linearity, precision, recovery, selectivity and sensitivity were found satisfactory for each analysed compound. Quantitation limits ranged from 26 to 240 fmol

  17. A preliminary X-ray study of transketolase from Burkholderia pseudomallei

    The transketolase TktA from B. pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were collected to 2.0 Å resolution. TktA is the most critical enzyme in the nonoxidative pentose phosphate pathway. It catalyzes the conversion of xylulose 5-phosphate and ribose 5-phosphate into sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate, and its products are used in the biosynthesis of acetyl-CoA, aromatic amino acids, nucleic acids and ADP-l-glycero-β-d-manno-heptose. TktA also has an unexpected role in chromosome structure that is independent of its metabolic responsibilities. Therefore, it is a new potent antibiotic target. In this study, TktA from Burkholderia pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.0 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 146.2, b = 74.6, c = 61.6 Å, β = 113.0°. A full structural determination is under way in order to provide insight into the structure–function relationship of this protein

  18. MR imaging signal enhancement of normal intracranial and extracranial structures

    The authors report their initial experience using a paramagnetic manganese chelate complex as a contrast agent for magnetic resonance (MR) imaging of the central nervous system. Five female cats weighing 2-4 kg were used, and anesthesia was induced and maintained with intravenous nembutal (15-25 mg/kg). This contrast agent, manganese (II) N, N'-bis(pyridoxal-5-phosphate) ethylenediamine-N,N'-diacetic acid, or Mn(DPDP)(Salutar), has previously shown efficacy for MR imaging of the hepatobiliary axis but has not been employed in neuroradiologic imaging. T1-weighted (repetition time, 400 msec, echo time, 15, 26 msec; 4-mm sections) spin-echo images were acquired before and after intravenous administration (100 μmol/kg) of the contrast agent. On post-contrast images, the pituitary gland, infundibulum and portohypophyseal system of the hypothalamus and choroid lexus demonstrated signal increase at t=0-30 minutes after injection

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

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

    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

  1. Vitamin B6s inhibit oxidative stress caused by Alzheimer's disease-related Cu(II)-β-amyloid complexes-cooperative action of phospho-moiety.

    Hashim, Alaa; Wang, Le; Juneja, Kashmir; Ye, Yong; Zhao, Yufen; Ming, Li-June

    2011-11-01

    Cu(II) complexes of Alzheimer's disease-related β-amyloid (Aβ) peptides exhibit metal-centered oxidation chemistry. The metallo-Aβ complexes are the hallmark of the disease and have been attributed to the generation of reactive oxygen species (ROS), causing oxidative stress. In this communication, the inhibitions of the oxidative activity of Cu(II)-Aβ by vitamin B6 compounds pyridoxamine (PM), pyridoxine (PN), pyridoxal (PL), and pyridoxal-5'-phosphate (PLP) are presented. These B6's are competitive inhibitors toward dopamine oxidation by Cu(II)-Aβ(1-20), with K(i) values of 1.4, 8.3, 1.2, and 0.2mM, respectively. The phospho-moiety in PLP seems to exhibit cooperative inhibition, affording a clue for future design of inhibitors. PMID:21944860

  2. A novel riboflavin gastro-mucoadhesive delivery system based on ion-exchange fiber.

    Yao, Huimin; Xu, Lu; Han, Fei; Che, Xin; Dong, Yang; Wei, Min; Guan, Jiao; Shi, Xiaolei; Li, Sanming

    2008-11-19

    A novel gastro-mucoadhesive delivery system based on ion-exchange fiber has been developed. Riboflavin-5'-phosphate sodium salt (RF5P), which is site-specifically absorbed from the upper gastrointestinal tract, was used as model drug. A modified dissolution system, which can also be called 'flow through diffusion cell' (FTDC), was used to study the drug release from the drug fibers. Gastrointestinal transit studies of the RF5P fiber complexes in rats and gamma imaging study in volunteer was carried out to evaluate the gastro-retentive behavior of the fiber. The pharmacokinetic profile and parameters of riboflavin via analysis of urinary excretion of riboflavin on man were measured. Study on rat and man provide evidence for the validity of the hypothesis that the drug fiber provided good mucoadhesive properties in vivo and should therefore be of considerable interest for the development of future mucoadhesive oral drug delivery dosage forms. PMID:18761065

  3. Template Directed Oligomer Ligation in Eutectic Phases in Water-Ice

    Dörr, Mark; Löffler, Philipp M. G.; Wieczorek, Rafal;

    2011-01-01

    Eutectic phases of water-ice are protective micro-environments for the non-enzymatic, metal ion mediated polymerization of imidazole-activated ribonucleotides (Monnard 2008). Polymers of up to 30-mer lengths can herein be achieved. Even longer polymers can be obtained by adding activated monomers...... or short oligomers to a solution over several freeze-thawing cycles. The final length is determined by the balancing rates of condensation and hydrolysis. A certain critical length of about 25 to 30 oligonucleotides is required to show catalytic activity (Scott 1995). This length can be much quicker....... (a) Reaction scheme of the condensation reaction of two oligoribonucleotides : The leaving group in this example is imidazole. (b) Illustration of a possible spatial arrangement of a template (15nt) directed ligation. The 7-mer is activated with imidazole at the 5' phosphate (apical moiety in the...

  4. The Measurement of PO43-, SO42- and NO3- anions ss the impurities of gadolinium and dysprosium purification

    The separation of phosphate, sulfate, and nitrate have been done by liquid chromatography, with eluent the mixture of sodium borate, sodium gluconate, butanol, and acetonitrile, and the conductivity was used as the detector. Separation was done by 1 part of butanol, and 6 parts of acetonitrile mixture as the eluent. The chromatogram showed that separation between nitrate-phosphate-sulfate had a good resolution, with 4.5 for nitrate-phosphate, and 6.7 for phosphate-sulfate. The regression standard were mode simultaneously, and the regression equations of nitrate Y = 1,24 + 3,5; phosphate: Y 1,75 X + 8,3; and sulfate: Y = 1,07 X + 4,6. Elution to the sample showed that the content of nitrate was too high, and overlap with the phosphate chromatogram, so that the content of phosphat in the sample could not to be measured. (author)

  5. Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?

    Rolland, N; Job, D; Douce, R

    1993-08-01

    A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5'-phosphate cofactor to be identified. The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution. The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor. The comparison also suggests that the 5'-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme. PMID:7916619

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

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

  7. Transaldolase Deficiency: Liver Cirrhosis Associated with a New Inborn Error in the Pentose Phosphate Pathway

    Verhoeven, Nanda M.; Huck, Jojanneke H. J.; Roos, Birthe; Struys, Eduard A.; Salomons, Gajja S.; Douwes, Adriaan C.; van der Knaap, Marjo S.; Jakobs, Cornelis

    2001-01-01

    This article describes the first patient with a deficiency of transaldolase (TALDO1 [E.C.2.2.1.2]). Clinically, the patient presented with liver cirrhosis and hepatosplenomegaly during early infancy. In urine and plasma, elevated concentrations of ribitol, d-arabitol, and erythritol were found. By incubating the patient's lymphoblasts and erythrocytes with ribose-5-phosphate and subsequently analyzing phosphate sugar metabolites, we discovered a deficiency of transaldolase. Sequence analysis of the transaldolase gene from this patient showed a homozygous deletion of 3 bp. This deletion results in absence of serine at position 171 of the transaldolase protein. This amino acid is invariable between species and is located in a conserved region, indicating its importance for enzyme activity. The detection of this new inborn error of pentose metabolism has implications for the diagnostic workup of liver problems of unknown etiology. PMID:11283793

  8. Uptake and expulsion of 14C-xylitol by xylitol-cultured Streptococcus mutans ATCC 25175 in vitro

    The effect of successive cultivations in the presence of 6% xylitol on the uptake and expulsion of 14C-xylitol was studied using the cells of Streptococcus mutans 25175. Three sequential cultivations did not alter the growth inhibition percentage (approximately 50%) observed in the presence of 6% xylitol. The 14C-xylitol uptake experiments performed with growing and resting cells showed that both the uptake and the expulsion of xylitol were enhanced by xylitolculturing. Both xylitol-cultured and resting control cells contained only one major labeled compound which was identified as 14C-xylitol 5-phosphate. The label subsequently was expelled from the cells as 14C-xylitol. These results indicate that S. mutans possesses an intracellular xylitol cycle and this cycle is regulated by adding xylitol to the growth medium. (author)

  9. Uptake and expulsion of sup 14 C-xylitol by xylitol-cultured Streptococcus mutans ATCC 25175 in vitro

    Soederling, E.; Pihlanto-Leppaelae, A. (Department of Biochemistry, Institute of Dentistry, University of Turku, Turku (Finland))

    1989-01-01

    The effect of successive cultivations in the presence of 6% xylitol on the uptake and expulsion of {sup 14}C-xylitol was studied using the cells of Streptococcus mutans 25175. Three sequential cultivations did not alter the growth inhibition percentage (approximately 50%) observed in the presence of 6% xylitol. The {sup 14}C-xylitol uptake experiments performed with growing and resting cells showed that both the uptake and the expulsion of xylitol were enhanced by xylitolculturing. Both xylitol-cultured and resting control cells contained only one major labeled compound which was identified as {sup 14}C-xylitol 5-phosphate. The label subsequently was expelled from the cells as {sup 14}C-xylitol. These results indicate that S. mutans possesses an intracellular xylitol cycle and this cycle is regulated by adding xylitol to the growth medium. (author).

  10. AcEST: DK949163 [AcEST

    Full Text Available >tr|Q0G1Y2|Q0G1Y2_9RHIZ Cobalamin (5`-phosphate) synthase protein OS=Fulvimarina pelagi HTCC2506 GN=FP2506_...g, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Gapped BLAST and PSI-BLAST: a n...dro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Gapped BLAST an...rotein 1... 32 3.7 sp|O82643|ZIP9_ARATH Zinc transporter 9 OS=Arabidopsis thaliana...NDKVAPSKDPGKKD 1565 >sp|O82643|ZIP9_ARATH Zinc transporter 9 OS=Arabidopsis thaliana GN=ZIP9 PE=2 SV=1 Lengt