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Peter Schonheit - One of the best experts on this subject based on the ideXlab platform.

  • key enzymes of the semiphosphorylative entner doudoroff Pathway in the haloarchaeon haloferax volcanii characterization of glucose dehydrogenase gluconate dehydratase and 2 keto 3 deoxy 6 phosphogluconate aldolase
    Journal of Bacteriology, 2016
    Co-Authors: Janmoritz Sutter, Ulrike Johnsen, Juliabeate Tastensen, Jorg Soppa, Peter Schonheit
    Abstract:

    ABSTRACT The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) Pathway. So far, the key enzymes of this Pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED Pathway for glucose degradation in H. volcanii. IMPORTANCE The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff Pathway in the haloarchaeon Haloferax volcanii. The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff Pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation Pathways in the domain Archaea.

  • the entner doudoroff Pathway is an overlooked glycolytic route in cyanobacteria and plants
    Proceedings of the National Academy of Sciences of the United States of America, 2016
    Co-Authors: Xi Chen, Peter Schonheit, Karoline Schreiber, Jens Appel, Alexander Makowka, Berit Fahnrich, Mayo Roettger, Mohammad R Hajirezaei, Frank D Sonnichsen, William Martin
    Abstract:

    Glucose degradation Pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden-Meyerhof-Parnas (EMP) Pathway] and the oxidative pentose phosphate (OPP) Pathway. However, we found that both possess a third, previously overlooked Pathway of glucose breakdown: the Entner-Doudoroff (ED) Pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP Pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED Pathway, was detected in both in vivo, indicating an active ED Pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation Pathways were knocked out indicate that the ED Pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED Pathway has lower protein costs and ATP yields than the EMP Pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED Pathway does not generate futile cycles in organisms that fix CO2 via the Calvin-Benson cycle.

  • metabolism of pentose sugars in the hyperthermophilic archaea sulfolobus solfataricus and sulfolobus acidocaldarius
    Journal of Biological Chemistry, 2010
    Co-Authors: Charlotte E M Nunn, Peter Schonheit, David W. Hough, Ulrike Johnsen, Uwe Sauer, Tobias Fuhrer, Michael J. Danson
    Abstract:

    We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff Pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous Pathway. On further investigation, the catalytic promiscuity of the first enzyme in this Pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the Pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this Pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar Pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

  • the nonphosphorylative entner doudoroff Pathway in the thermoacidophilic euryarchaeon picrophilus torridus involves a novel 2 keto 3 deoxygluconate specific aldolase
    Journal of Bacteriology, 2010
    Co-Authors: Matthias Reher, Michael Bott, Tobias Fuhrer, Peter Schonheit
    Abstract:

    The Pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-13C]- and [3-13C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type Pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED Pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED Pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED Pathway involving a bifunctional KD(P)GA as a key enzyme.

  • characterization of glycerate kinase 2 phosphoglycerate forming a key enzyme of the nonphosphorylative entner doudoroff Pathway from the thermoacidophilic euryarchaeon picrophilus torridus
    Fems Microbiology Letters, 2006
    Co-Authors: Matthias Reher, Michael Bott, Peter Schonheit
    Abstract:

    Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Entner–Doudoroff (ED) Pathway. Here we report the characterization of a key enzyme of this Pathway, glycerate kinase (2-phosphoglycerate forming). The enzyme was purified 5100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to 2-phosphoglycerate. The enzyme showed highest activity at 60°C and pH 7.3, with ATP as phosphoryl donor and Mg2+ as divalent cation. By MALDI-TOF analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus tenax, for which the operation of nonphosphorylative ED Pathways, involving 2-phosphoglycerate forming glycerate kinases, has been proposed.

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

  • Insights into the Substrate Specificity of Archaeal Entner–Doudoroff Aldolases: The Structures of Picrophilus torridus 2‑Keto-3-deoxygluconate Aldolase and Sulfolobus solfataricus 2‑Keto-3-deoxy-6-phosphogluconate Aldolase in Complex with 2‑Keto-3-de
    2018
    Co-Authors: Viatcheslav Zaitsev, Michael J. Danson, Matthias Reher, Garry L. Taylor, Ulrike Johnsen, Marius Ortjohann, Peter Schönheit, Susan J. Crennell
    Abstract:

    The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolize glucose via a nonphosphorylative Entner–Doudoroff Pathway and a branched Entner–Doudoroff Pathway, respectively. Key enzymes for these Entner–Doudoroff Pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase [KD­(P)­G-aldolase]. KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the nonphosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD­(P)­G-aldolase from S. solfataricus [Ss-KD­(P)­G-aldolase] is an enzyme that catalyzes the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD­(P)­G-aldolase has not been analyzed before. In this work, we report the elucidation of the structure of Ss-KD­(P)­G-aldolase in complex with KDPG at 2.35 Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely, Arg106, Tyr132, Arg237, and Ser241, was identified in Ss-KD­(P)­G-aldolase that interact with the negatively charged phosphate group of KDPG, thereby increasing the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage

  • metabolism of pentose sugars in the hyperthermophilic archaea sulfolobus solfataricus and sulfolobus acidocaldarius
    Journal of Biological Chemistry, 2010
    Co-Authors: Charlotte E M Nunn, Peter Schonheit, David W. Hough, Ulrike Johnsen, Uwe Sauer, Tobias Fuhrer, Michael J. Danson
    Abstract:

    We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff Pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous Pathway. On further investigation, the catalytic promiscuity of the first enzyme in this Pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the Pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this Pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar Pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

  • the structural basis for substrate promiscuity in 2 keto 3 deoxygluconate aldolase from the entner doudoroff Pathway in sulfolobus solfataricus
    Journal of Biological Chemistry, 2004
    Co-Authors: Alex Theodossis, Michael J. Danson, David W. Hough, Henry J Lamble, Helen Walden, Elaine J Westwick, Helen Connaris, Garry L. Taylor
    Abstract:

    Abstract The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 °C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff Pathway. In this Pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to d-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to d-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this Pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic Pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and d-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and d-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and d-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 °C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus d-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.

  • gluconate dehydratase from the promiscuous entner doudoroff Pathway in sulfolobus solfataricus
    FEBS Letters, 2004
    Co-Authors: Henry J Lamble, Garry L. Taylor, David W. Hough, Christine C Milburn, Michael J. Danson
    Abstract:

    An investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 °C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner–Doudoroff Pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose.

  • metabolic Pathway promiscuity in the archaeon sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2 keto 3 deoxygluconate aldolase
    Journal of Biological Chemistry, 2003
    Co-Authors: Henry J Lamble, David W. Hough, Narinder I Heyer, Steven D Bull, Michael J. Danson
    Abstract:

    The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff Pathway. In this Pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.

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

  • Metabolic engineering of Gluconobacter oxydans 621H for increased biomass yield.
    Applied Microbiology and Biotechnology, 2017
    Co-Authors: Ines Kiefler, Stephanie Bringer, Michael Bott
    Abstract:

    The obligatory aerobic acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources regio- and stereoselectively in the periplasm and therefore is used industrially for oxidative biotransformations, e. g., in vitamin C production. However, it has a very low biomass yield as the oxidized products largely remain in the medium and cannot be used for anabolism. Cytoplasmic carbon metabolism occurs via the pentose phosphate Pathway and the Entner-Doudoroff Pathway, whereas glycolysis and the tricarboxylic acid cycle are incomplete. Acetate is formed as an end product via pyruvate decarboxylase and acetaldehyde dehydrogenase. In order to increase the biomass yield from glucose, we sequentially replaced (i) gdhS encoding the cytoplasmic NADP-dependent glucose dehydrogenase by the Acetobacter pasteurianus sdhCDABE genes for succinate dehydrogenase and the flavinylation factor SdhE (strain IK001), (ii) pdc encoding pyruvate decarboxylase by a second ndh gene encoding a type II NADH dehydrogenase (strain IK002.1), and (iii) gdhM encoding the membrane-bound PQQ-dependent glucose dehydrogenase by sucCD from Gluconacetobacter diazotrophicus encoding succinyl-CoA synthetase (strain IK003.1). Analysis of the strains under controlled cultivation conditions in bioreactors revealed for IK003.1 that neither gluconate nor 2-ketogluconate was formed, but some 5-ketogluconate. Acetate formation was eliminated, and comparable amounts of pyruvate were formed instead. CO2 formation by IK003.1 was more than doubled compared to the reference strain. Growth of IK003.1 was retarded, but the biomass yield of this strain was raised by 60%. IK003.1 serves as suitable host for oxidative biotransformations and for further metabolic engineering.

  • role of the pentose phosphate Pathway and the entner doudoroff Pathway in glucose metabolism of gluconobacter oxydans 621h
    Applied Microbiology and Biotechnology, 2013
    Co-Authors: Janine Richhardt, Stephanie Bringer, Michael Bott
    Abstract:

    Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate Pathway (PPP) and the Entner-Doudoroff Pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd-eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two Pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.

  • the nonphosphorylative entner doudoroff Pathway in the thermoacidophilic euryarchaeon picrophilus torridus involves a novel 2 keto 3 deoxygluconate specific aldolase
    Journal of Bacteriology, 2010
    Co-Authors: Matthias Reher, Michael Bott, Tobias Fuhrer, Peter Schonheit
    Abstract:

    The Pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-13C]- and [3-13C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type Pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED Pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED Pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED Pathway involving a bifunctional KD(P)GA as a key enzyme.

  • characterization of glycerate kinase 2 phosphoglycerate forming a key enzyme of the nonphosphorylative entner doudoroff Pathway from the thermoacidophilic euryarchaeon picrophilus torridus
    Fems Microbiology Letters, 2006
    Co-Authors: Matthias Reher, Michael Bott, Peter Schonheit
    Abstract:

    Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Entner–Doudoroff (ED) Pathway. Here we report the characterization of a key enzyme of this Pathway, glycerate kinase (2-phosphoglycerate forming). The enzyme was purified 5100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to 2-phosphoglycerate. The enzyme showed highest activity at 60°C and pH 7.3, with ATP as phosphoryl donor and Mg2+ as divalent cation. By MALDI-TOF analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus tenax, for which the operation of nonphosphorylative ED Pathways, involving 2-phosphoglycerate forming glycerate kinases, has been proposed.

Xueming Zhao - One of the best experts on this subject based on the ideXlab platform.

  • RESEARCH Open Access Engineering of Serine-Deamination Pathway,
    2016
    Co-Authors: Tao Chen, Xueming Zhao
    Abstract:

    Entner-Doudoroff Pathway Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose o

  • engineering of serine deamination Pathway entner doudoroff Pathway and pyruvate dehydrogenase complex to improve poly 3 hydroxybutyrate production in escherichia coli
    Microbial Cell Factories, 2014
    Co-Authors: Yan Zhang, Zhenquan Lin, Qiaojie Liu, Zhiwen Wang, Tao Chen, Xueming Zhao
    Abstract:

    Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources. In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) Pathway, the Entner-Doudoroff (ED) Pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED Pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio were enhanced after the modification of SD Pathway, ED Pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source. Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination Pathway, Entner-Doudoroff Pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

  • engineering of serine deamination Pathway entner doudoroff Pathway and pyruvate dehydrogenase complex to improve poly 3 hydroxybutyrate production in escherichia coli
    Microbial Cell Factories, 2014
    Co-Authors: Yan Zhang, Tao Chen, Yifan Li, Zhongqiang Wang, Xueming Zhao
    Abstract:

    Background: Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources. Results: In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) Pathway, the Entner-Doudoroff (ED) Pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha ,p roduced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED Pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP + ] ratio were enhanced after the modification of SD Pathway, ED Pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source. Conclusions: Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination Pathway, Entner-Doudoroff Pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

David W. Hough - One of the best experts on this subject based on the ideXlab platform.

  • metabolism of pentose sugars in the hyperthermophilic archaea sulfolobus solfataricus and sulfolobus acidocaldarius
    Journal of Biological Chemistry, 2010
    Co-Authors: Charlotte E M Nunn, Peter Schonheit, David W. Hough, Ulrike Johnsen, Uwe Sauer, Tobias Fuhrer, Michael J. Danson
    Abstract:

    We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff Pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous Pathway. On further investigation, the catalytic promiscuity of the first enzyme in this Pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the Pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this Pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar Pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

  • the structural basis for substrate promiscuity in 2 keto 3 deoxygluconate aldolase from the entner doudoroff Pathway in sulfolobus solfataricus
    Journal of Biological Chemistry, 2004
    Co-Authors: Alex Theodossis, Michael J. Danson, David W. Hough, Henry J Lamble, Helen Walden, Elaine J Westwick, Helen Connaris, Garry L. Taylor
    Abstract:

    Abstract The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 °C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff Pathway. In this Pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to d-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to d-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this Pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic Pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and d-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and d-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and d-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 °C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus d-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.

  • gluconate dehydratase from the promiscuous entner doudoroff Pathway in sulfolobus solfataricus
    FEBS Letters, 2004
    Co-Authors: Henry J Lamble, Garry L. Taylor, David W. Hough, Christine C Milburn, Michael J. Danson
    Abstract:

    An investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 °C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner–Doudoroff Pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose.

  • metabolic Pathway promiscuity in the archaeon sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2 keto 3 deoxygluconate aldolase
    Journal of Biological Chemistry, 2003
    Co-Authors: Henry J Lamble, David W. Hough, Narinder I Heyer, Steven D Bull, Michael J. Danson
    Abstract:

    The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff Pathway. In this Pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.

  • an extremely thermostable aldolase from sulfolobus solfataricus with specificity for non phosphorylated substrates
    Biochemical Journal, 1999
    Co-Authors: Catriona L Buchanan, Michael J. Danson, Helen Connaris, Christopher D Reeve, David W. Hough
    Abstract:

    Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff Pathway, in which the reversible C(6) to C(3) aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S. solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme. The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S. solfataricus cells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equally active with both d- and l-glyceraldehyde. It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH(4).