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Winfried Boos - One of the best experts on this subject based on the ideXlab platform.

  • tret a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Biological Chemistry, 2004
    Co-Authors: Qiuhao Qu, Winfried Boos
    Abstract:

    Abstract The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 °C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the Vmax is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the Vmax was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium.

  • trmb a sugar specific transcriptional regulator of the trehalose maltose abc transporter from the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Biological Chemistry, 2003
    Co-Authors: Afra Engelmann, Reinhold Horlacher, Gudrun Vierke, Carina Hebbeln, Qiuhao Qu, Michael Thomm, Winfried Boos
    Abstract:

    Abstract We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 μm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 °C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  • trmb a sugar specific transcriptional regulator of the trehalose maltose abc transporter from the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Biological Chemistry, 2003
    Co-Authors: Afra Engelmann, Reinhold Horlacher, Gudrun Vierke, Carina Hebbeln, Qiuhao Qu, Michael Thomm, Winfried Boos
    Abstract:

    Abstract We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 μm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 °C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  • crystallization and preliminary x ray analysis of the trehalose maltose abc transporter malfgk2 from Thermococcus litoralis
    Acta Crystallographica Section D-biological Crystallography, 2002
    Co-Authors: André Schiefner, Winfried Boos, Kay Diederichs, K. Hashimoto, Wolfram Welte
    Abstract:

    Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

  • Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis.
    Acta Crystallographica Section D Biological Crystallography, 2002
    Co-Authors: André Schiefner, Winfried Boos, Kay Diederichs, K. Hashimoto, Wolfram Welte
    Abstract:

    Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

Reinhold Horlacher - One of the best experts on this subject based on the ideXlab platform.

  • trmb a sugar specific transcriptional regulator of the trehalose maltose abc transporter from the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Biological Chemistry, 2003
    Co-Authors: Afra Engelmann, Reinhold Horlacher, Gudrun Vierke, Carina Hebbeln, Qiuhao Qu, Michael Thomm, Winfried Boos
    Abstract:

    Abstract We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 μm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 °C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  • trmb a sugar specific transcriptional regulator of the trehalose maltose abc transporter from the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Biological Chemistry, 2003
    Co-Authors: Afra Engelmann, Reinhold Horlacher, Gudrun Vierke, Carina Hebbeln, Qiuhao Qu, Michael Thomm, Winfried Boos
    Abstract:

    Abstract We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 μm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 °C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  • TrmB, a Sugar-specific Transcriptional Regulator of the Trehalose/Maltose ABC Transporter from the Hyperthermophilic Archaeon Thermococcus litoralis
    The Journal of biological chemistry, 2002
    Co-Authors: Sung-jae Lee, Afra Engelmann, Reinhold Horlacher, Gudrun Vierke, Carina Hebbeln, Michael Thomm, Winfried Boos
    Abstract:

    Abstract We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 μm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 °C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  • the crystal structure of a liganded trehalose maltose binding protein from the hyperthermophilic archaeon Thermococcus litoralis at 1 85 a
    Journal of Molecular Biology, 2001
    Co-Authors: Joachim Diez, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, Wolfram Welte
    Abstract:

    Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

  • The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.
    Journal of molecular biology, 2001
    Co-Authors: Joachim Diez, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, Wolfram Welte
    Abstract:

    Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Wolfram Welte - One of the best experts on this subject based on the ideXlab platform.

  • crystallization and preliminary x ray analysis of the trehalose maltose abc transporter malfgk2 from Thermococcus litoralis
    Acta Crystallographica Section D-biological Crystallography, 2002
    Co-Authors: André Schiefner, Winfried Boos, Kay Diederichs, K. Hashimoto, Wolfram Welte
    Abstract:

    Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

  • Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis.
    Acta Crystallographica Section D Biological Crystallography, 2002
    Co-Authors: André Schiefner, Winfried Boos, Kay Diederichs, K. Hashimoto, Wolfram Welte
    Abstract:

    Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

  • the crystal structure of a liganded trehalose maltose binding protein from the hyperthermophilic archaeon Thermococcus litoralis at 1 85 a
    Journal of Molecular Biology, 2001
    Co-Authors: Joachim Diez, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, Wolfram Welte
    Abstract:

    Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

  • The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.
    Journal of molecular biology, 2001
    Co-Authors: Joachim Diez, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, Wolfram Welte
    Abstract:

    Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

  • the crystal structure of a liganded trehalose maltose binding protein from the hyperthermophilic archaeon Thermococcus litoralis at 1 85 a
    Journal of Molecular Biology, 2001
    Co-Authors: Joachim Diez, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, Wolfram Welte
    Abstract:

    Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Helena Santos - One of the best experts on this subject based on the ideXlab platform.

  • maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis purification and characterization of key enzymes
    Journal of Bacteriology, 1999
    Co-Authors: Karina B Xavier, Winfried Boos, Ralf Peist, M Kossmann, Helena Santos
    Abstract:

    Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

  • archaeal binding protein dependent abc transporter molecular and biochemical analysis of the trehalose maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Bacteriology, 1998
    Co-Authors: Reinhold Horlacher, Karina B Xavier, M Kossmann, Helena Santos, Jocelyne Diruggiero, Winfried Boos
    Abstract:

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

  • Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis
    Journal of bacteriology, 1998
    Co-Authors: Reinhold Horlacher, Karina B Xavier, M Kossmann, Helena Santos, Jocelyne Diruggiero, Winfried Boos
    Abstract:

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

  • archaeal binding protein dependent abc transporter molecular and biochemical analysis of the trehalose maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Bacteriology, 1998
    Co-Authors: Reinhold Horlacher, Karina B Xavier, M Kossmann, Helena Santos, Jocelyne Diruggiero, Winfried Boos
    Abstract:

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/ maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a Kd of 0.16 mM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches. High-affinity binding protein-dependent ABC transporters were originally discovered in gram-negative bacteria. They consist of a high-affinity substrate-binding protein located in the periplasmic space as their major substrate recognition site, two hydrophobic membrane proteins forming the translocation pore, and two additional subunits peripherally associated with the membrane proteins at the inner face of the membrane. By ATP hydrolysis the last two subunits provide the energy for the accumulation of substrate against the concentration gradient (7). In the case of the Escherichia coli maltose/maltodextrin transport system, the periplasmic binding protein (maltosebinding protein or MalE) is encoded by malE, the membrane components MalF and MalG are encoded by the malF and malG genes, and the two ATP-hydrolyzing subunits of MalK are encoded by malK. These genes form a cluster on the E. coli chromosome in which malE, malF, and malG constitute an operon that is oriented divergently to malK (8). Recently, it has been recognized that binding protein-dependent ABC transporters are also present in gram-positive bacteria (20). In these cases, the soluble periplasmic binding proteins are anchored in the membrane by an N-terminal lipid modification consisting of a diglyceride connected to the N-terminal cysteine via a thioether bond (51). Binding protein-dependent ABC transporters have also been found in thermophilic bacteria (25, 41). Despite the large amount of information available on this type of transport system in bacteria, only one study of an archaeal ABC system, that of the hyperthermophile Thermococcus litoralis, has been reported so far (52). This transport system has several unusual properties: it shows an extremely high affinity (Km of about 20 nM) at 85°C, the optimum growth temperature of this organism; it recognizes with equal affinity its very different substrates, maltose and trehalose; and it is not inhibited by maltodextrins. We undertook to further characterize this newly discovered transport system. Here we report on the purification of the native trehalose/maltose-binding protein (TMBP), the cloning and sequencing of the malEFG gene cluster, and the expression of the malE gene in E. coli as well as the purification and characterization of its encoded binding protein. The rationale for analyzing a binding protein-dependent transport system from a hyperthermophilic organism whose function is optimal at 85°C but is less than 5% at room temperature is the expectation that is conformation will be more rigid at room temperature and will become accessible to structural analysis under these conditions. In addition, evolutionary aspects and its unusual substrate specificity make it attractive for study.

  • high affinity maltose trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Bacteriology, 1996
    Co-Authors: Karina B Xavier, Winfried Boos, Ligia O Martins, Ralf Peist, M Kossmann, Helena Santos
    Abstract:

    The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

Karina B Xavier - One of the best experts on this subject based on the ideXlab platform.

  • maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis purification and characterization of key enzymes
    Journal of Bacteriology, 1999
    Co-Authors: Karina B Xavier, Winfried Boos, Ralf Peist, M Kossmann, Helena Santos
    Abstract:

    Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

  • archaeal binding protein dependent abc transporter molecular and biochemical analysis of the trehalose maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Bacteriology, 1998
    Co-Authors: Reinhold Horlacher, Karina B Xavier, M Kossmann, Helena Santos, Jocelyne Diruggiero, Winfried Boos
    Abstract:

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

  • Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis
    Journal of bacteriology, 1998
    Co-Authors: Reinhold Horlacher, Karina B Xavier, M Kossmann, Helena Santos, Jocelyne Diruggiero, Winfried Boos
    Abstract:

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

  • archaeal binding protein dependent abc transporter molecular and biochemical analysis of the trehalose maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Bacteriology, 1998
    Co-Authors: Reinhold Horlacher, Karina B Xavier, M Kossmann, Helena Santos, Jocelyne Diruggiero, Winfried Boos
    Abstract:

    We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/ maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a Kd of 0.16 mM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches. High-affinity binding protein-dependent ABC transporters were originally discovered in gram-negative bacteria. They consist of a high-affinity substrate-binding protein located in the periplasmic space as their major substrate recognition site, two hydrophobic membrane proteins forming the translocation pore, and two additional subunits peripherally associated with the membrane proteins at the inner face of the membrane. By ATP hydrolysis the last two subunits provide the energy for the accumulation of substrate against the concentration gradient (7). In the case of the Escherichia coli maltose/maltodextrin transport system, the periplasmic binding protein (maltosebinding protein or MalE) is encoded by malE, the membrane components MalF and MalG are encoded by the malF and malG genes, and the two ATP-hydrolyzing subunits of MalK are encoded by malK. These genes form a cluster on the E. coli chromosome in which malE, malF, and malG constitute an operon that is oriented divergently to malK (8). Recently, it has been recognized that binding protein-dependent ABC transporters are also present in gram-positive bacteria (20). In these cases, the soluble periplasmic binding proteins are anchored in the membrane by an N-terminal lipid modification consisting of a diglyceride connected to the N-terminal cysteine via a thioether bond (51). Binding protein-dependent ABC transporters have also been found in thermophilic bacteria (25, 41). Despite the large amount of information available on this type of transport system in bacteria, only one study of an archaeal ABC system, that of the hyperthermophile Thermococcus litoralis, has been reported so far (52). This transport system has several unusual properties: it shows an extremely high affinity (Km of about 20 nM) at 85°C, the optimum growth temperature of this organism; it recognizes with equal affinity its very different substrates, maltose and trehalose; and it is not inhibited by maltodextrins. We undertook to further characterize this newly discovered transport system. Here we report on the purification of the native trehalose/maltose-binding protein (TMBP), the cloning and sequencing of the malEFG gene cluster, and the expression of the malE gene in E. coli as well as the purification and characterization of its encoded binding protein. The rationale for analyzing a binding protein-dependent transport system from a hyperthermophilic organism whose function is optimal at 85°C but is less than 5% at room temperature is the expectation that is conformation will be more rigid at room temperature and will become accessible to structural analysis under these conditions. In addition, evolutionary aspects and its unusual substrate specificity make it attractive for study.

  • high affinity maltose trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis
    Journal of Bacteriology, 1996
    Co-Authors: Karina B Xavier, Winfried Boos, Ligia O Martins, Ralf Peist, M Kossmann, Helena Santos
    Abstract:

    The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.