Xylulose 5-Phosphate

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Michael Teuber - One of the best experts on this subject based on the ideXlab platform.

  • characterization of the d Xylulose 5 phosphate d fructose 6 phosphate phosphoketolase gene xfp from bifidobacterium lactis
    Journal of Bacteriology, 2001
    Co-Authors: Leo Meile, L. M. Rohr, T. A. Geissmann, M. Herensperger, Michael Teuber
    Abstract:

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

  • Characterization of the D-Xylulose 5-Phosphate/D-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis
    Journal of Bacteriology, 2001
    Co-Authors: Leo Meile, L. M. Rohr, T. A. Geissmann, M. Herensperger, Michael Teuber
    Abstract:

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

Kerry S. Smith - One of the best experts on this subject based on the ideXlab platform.

  • allosteric regulation of lactobacillus plantarum Xylulose 5 phosphate fructose 6 phosphate phosphoketolase xfp
    Journal of Bacteriology, 2015
    Co-Authors: Katie Glenn, Kerry S. Smith
    Abstract:

    ABSTRACT Xylulose 5-Phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of Xylulose 5-Phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and P i ) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-Phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.

  • biochemical and kinetic characterization of Xylulose 5 phosphate fructose 6 phosphate phosphoketolase 2 xfp2 from cryptococcus neoformans
    Eukaryotic Cell, 2014
    Co-Authors: Katie Glenn, Cheryl Ingramsmith, Kerry S. Smith
    Abstract:

    Xylulose 5-Phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from Xylulose 5-Phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC50) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.

Leo Meile - One of the best experts on this subject based on the ideXlab platform.

  • characterization of the d Xylulose 5 phosphate d fructose 6 phosphate phosphoketolase gene xfp from bifidobacterium lactis
    Journal of Bacteriology, 2001
    Co-Authors: Leo Meile, L. M. Rohr, T. A. Geissmann, M. Herensperger, Michael Teuber
    Abstract:

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

  • Characterization of the D-Xylulose 5-Phosphate/D-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis
    Journal of Bacteriology, 2001
    Co-Authors: Leo Meile, L. M. Rohr, T. A. Geissmann, M. Herensperger, Michael Teuber
    Abstract:

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

Tomohisa Kuzuyama - One of the best experts on this subject based on the ideXlab platform.

  • antimicrobial n 2 chlorobenzyl substituted hydroxamate is an inhibitor of 1 deoxy d Xylulose 5 phosphate synthase
    Chemical Communications, 2013
    Co-Authors: Daisuke Hayashi, Tomohisa Kuzuyama, Nobuo Kato, Yasuo Sato, Junko Ohkanda
    Abstract:

    N-(2-Chlorobenzyl)-substituted hydroxamate, readily produced by hydrolysis of ketoclomazone, was identified as an inhibitor of 1-deoxy-D-Xylulose 5-Phosphate synthase (DXS), with an IC50 value of 1.0 μM. The compound inhibited the growth of Haemophilus influenzae. A convenient spectroscopic method for assaying DXS using NADPH–lactate dehydrogenase (LDH) is also reported.

  • the single cellular green microalga botryococcus braunii race b possesses three distinct 1 deoxy d Xylulose 5 phosphate synthases
    Plant Science, 2012
    Co-Authors: Daisuke Matsushima, Tomohisa Kuzuyama, Holger Jenkekodama, Yohei Sato, Yusuke Fukunaga, Koremitsu Sumimoto, Shigeki Matsunaga, Shigeru Okada
    Abstract:

    Abstract Green algae exclusively use the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isoprenoids. The first enzyme of this pathway is 1-deoxy- d -Xylulose 5-Phosphate synthase (DXS, EC 2.2.1.7). Green algae have been thought to possess only a single DXS, in contrast to land plants, which have at least two isoforms that serve different roles in metabolism. The green microalga Botryococcus braunii has an extraordinary isoprenoid metabolism, as it produces large amounts of triterpene hydrocarbons. Here, we did cDNA cloning of DXSs from B. braunii and examined enzyme activities of the heterologously expressed proteins. Three distinct DXS isoforms were identified, all of which were functional and had similar kinetic properties, whereas the temperature dependence of enzyme activity showed considerable differences. Transcription of the genes was examined by real time quantitative RT-PCR. The three DXS genes were simultaneously expressed, and the expression levels were highest on day six after subculturing. B. braunii is the first green microalga demonstrated to have multiple DXS isoforms like land plants. This difference to other microalgae seems to mirror its special needs for extensive triterpene production by increasing the metabolic flow through the MEP pathway.

  • structure of 1 deoxy d Xylulose 5 phosphate reductoisomerase in a quaternary complex with a magnesium ion nadph and the antimalarial drug fosmidomycin
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007
    Co-Authors: Shunsuke Yajima, Tomohisa Kuzuyama, Kanju Ohsawa, Kodai Hara, Daisuke Iino, Yasuyuki Sasaki, Haruo Seto
    Abstract:

    The crystal structure of 1-deoxy-d-Xylulose 5-Phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg2+, NADPH and fosmidomycin was solved at 2.2 A resolution. DXR is the key enzyme in the 2-­C-methyl-d-­erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg2+ was shifted by NADPH in the active site.

  • identification of class 2 1 deoxy d Xylulose 5 phosphate synthase and 1 deoxy d Xylulose 5 phosphate reductoisomerase genes from ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis
    Planta Medica, 2006
    Co-Authors: Sangmin Kim, Tomohisa Kuzuyama, Yungjin Chang, Kwangseop Song, Sooun Kim
    Abstract:

    Diterpenoid ginkgolides having potent platelet-activating factor antagonist activity are major active ingredients of ginkgo extract. Class 2-type 1-deoxy-D-Xylulose 5-Phosphate synthase (GbDXS2) and 1-deoxy-D-Xylulose 5-Phosphate reductoisomerase (GbDXR), the first two enzymes in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, operating in the earlier step of ginkgolide biosynthesis, were cloned from embryonic roots of Ginkgo biloba through a homology-based polymerase chain reaction for role assessment of the enzymes. Plasmids harboring each gene rescued the respective knockout E. coli mutants. The levopimaradiene synthase gene (LPS), responsible for the first committed step in ginkgolide biosynthesis, and GbDXS2 were transcribed exclusively in embryonic root, suggesting a specific role of GbDXS2 in ginkgolide biosynthesis. GbDXR retained a higher transcription level in roots than in leaves, whereas class 1 DXS (GbDXS1) showed 30 to 50 % higher level in leaves. Ginkgolides and bilobalide were found both in leaves and roots from an earlier stage of the embryo culture. Exclusive transcription of ginkgolide biosynthesis-specific LPS and GbDXS2 in roots and the appearance of ginkgolides in leaves was consistent with translocation of the compounds from roots to leaves.

  • crystal structure of 1 deoxy d Xylulose 5 phosphate reductoisomerase complexed with cofactors implications of a flexible loop movement upon substrate binding
    Journal of Biochemistry, 2002
    Co-Authors: Shunsuke Yajima, Takamasa Nonaka, Tomohisa Kuzuyama, Haruo Seto, Kanju Ohsawa
    Abstract:

    The key enzyme in the nonmevalonate pathway, 1-deoxy-D-Xylulose 5-Phosphate reductoisomerase (DXR), has been shown to be an effective target of antimalarial drugs. Here we report the crystal structure of DXR complexed with NADPH and a sulfate ion from Escherichia coli at 2.2 A resolution. The structure showed the presence of an extra domain, which is absent from other NADPH-dependent oxidoreductases, in addition to the conformation of catalytic residues and the substrate binding site. A flexible loop covering the substrate binding site plays an important role in the enzymatic reaction and the determination of substrate specificity.

Frederick M Hahn - One of the best experts on this subject based on the ideXlab platform.

  • 1 deoxy d Xylulose 5 phosphate synthase the gene product of open reading frame orf 2816 and orf 2895 in rhodobacter capsulatus
    Journal of Bacteriology, 2001
    Co-Authors: Frederick M Hahn, Lisa M Eubanks, Charles A Testa, Brian S J Blagg, Jonathan A Baker, Dale C Poulter
    Abstract:

    In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-Xylulose 5-Phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-Xylulose 5-Phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-Xylulose 5-Phosphate synthase A and B, containing a His6 tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-Xylulose 5-Phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-Xylulose 5-Phosphate synthase A and B: Km pyruvate 5 0.61 and 3.0 mM, Km D-glyceraldehyde 3-phosphate 5 150 and 120 mM, and Vmax 5 1.9 and 1.4 mmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-DXylulose 5-Phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11. Isoprenoid compounds form a large, ubiquitous class of natural products consisting of over 30,000 individual members. They have a wide variety of cellular functions—such as components of cell membranes (sterols), electron transport (ubiquinones), signal transduction (prenylated proteins), photo

  • 1 deoxy d Xylulose 5 phosphate synthase the gene product of open reading frame orf 2816 and orf 2895 in rhodobacter capsulatus
    Journal of Bacteriology, 2001
    Co-Authors: Frederick M Hahn, Lisa M Eubanks, Charles A Testa, Brian S J Blagg, Jonathan A Baker, C D Poulter
    Abstract:

    In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-Xylulose 5-Phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-Xylulose 5-Phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-Xylulose 5-Phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-Xylulose 5-Phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-Xylulose 5-Phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-Xylulose 5-Phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.