6 Phosphofructokinase

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Sumiko Inouye - One of the best experts on this subject based on the ideXlab platform.

  • modulating factors for the pkn4 kinase cascade in regulating 6 Phosphofructokinase in myxococcus xanthus
    Molecular Microbiology, 2005
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus, a Gram-negative developmental bacterium, contains a large number of protein Ser/Thr kinases (PSTKs). Among these PSTKs, Pkn4 has been shown to be 6-Phosphofructokinase (PFK) kinase. PFK associates with the regulatory domain of Pkn4 (Pkn4RD) and is activated by Pkn4-mediated phosphorylation. The activation of PFK is required to consume glycogen accumulated during early development and is essential for efficient sporulation. Using the yeast two-hybrid screen, we identified three new factors, MkapA, MkapB and MkapC, that interact with Pkn4 and each contains well-known protein–protein interaction domains. MkapB contains eight tandem repeats of the TPR (tetratrico peptide repeat) domain and its interaction with Pkn4RD was phosphorylation-dependent. MkapB remained associated with Pkn4RD. As a result, Pkn4 did not interact with PFK and its activation was inhibited. While deletion of the pfk-pkn4 operon did not inhibit fruiting body formation, the spore yield was low. In contrast, a mkapB deletion mutant exhibited a 24 h delay in fruiting body formation, accumulated less glycogen in the stationary phase and gave rise to 3.2% spore formation as opposed to 100% attained with DZF1. In addition to Pkn4, MkapA associated with other membrane-associated PSTKs, Pkn1, Pkn2, Pkn8 and Pkn9, while MkapB associated with Pkn8 and Pkn9, and MkapC with Pkn8. These results indicate that there are complex PSTK networks in M. xanthus that share common modulating factors.

  • An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4-activated 6-Phosphofructokinase.
    Molecular microbiology, 2003
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary 6-Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7-fold upon phosphorylation at Thr-226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk–pkn4 deletion strain accumulated glycogen at a higher level than the wild-type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk–pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr-226-Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.

  • Activation of 6-Phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus.
    Molecular microbiology, 2002
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-Phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.

  • activation of 6 Phosphofructokinase via phosphorylation by pkn4 a protein ser thr kinase of myxococcus xanthus
    Molecular Microbiology, 2002
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-Phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.

Stephen B. Smith - One of the best experts on this subject based on the ideXlab platform.

  • postmortem regulation of glycolysis by 6 Phosphofructokinase in bovine m sternocephalicus pars mandibularis
    Meat Science, 2005
    Co-Authors: Ryan D. Rhoades, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Andy D King, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6-Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P<0.001) from 0.84μmol/g at d 0 to 6.54μmol/g at d 4. Fructose-6-phosphate and glucose-6-phosphate increased (P<0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33μmol/g) and was elevated to 45.9μmol/g by d 4. In the second experiment, conversion of [U-(14)C]glucose to lactate, glycogen, and CO(2) was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U-(14)C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123nmol glucose utilized per 100mg muscle per h; P=0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO(2) in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.

  • Postmortem regulation of glycolysis by 6-Phosphofructokinase in bovine M. Sternocephalicus pars mandibularis
    Meat science, 2005
    Co-Authors: Ryan D. Rhoades, D. Andy King, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6-Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P

Hirofumi Nariya - One of the best experts on this subject based on the ideXlab platform.

  • modulating factors for the pkn4 kinase cascade in regulating 6 Phosphofructokinase in myxococcus xanthus
    Molecular Microbiology, 2005
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus, a Gram-negative developmental bacterium, contains a large number of protein Ser/Thr kinases (PSTKs). Among these PSTKs, Pkn4 has been shown to be 6-Phosphofructokinase (PFK) kinase. PFK associates with the regulatory domain of Pkn4 (Pkn4RD) and is activated by Pkn4-mediated phosphorylation. The activation of PFK is required to consume glycogen accumulated during early development and is essential for efficient sporulation. Using the yeast two-hybrid screen, we identified three new factors, MkapA, MkapB and MkapC, that interact with Pkn4 and each contains well-known protein–protein interaction domains. MkapB contains eight tandem repeats of the TPR (tetratrico peptide repeat) domain and its interaction with Pkn4RD was phosphorylation-dependent. MkapB remained associated with Pkn4RD. As a result, Pkn4 did not interact with PFK and its activation was inhibited. While deletion of the pfk-pkn4 operon did not inhibit fruiting body formation, the spore yield was low. In contrast, a mkapB deletion mutant exhibited a 24 h delay in fruiting body formation, accumulated less glycogen in the stationary phase and gave rise to 3.2% spore formation as opposed to 100% attained with DZF1. In addition to Pkn4, MkapA associated with other membrane-associated PSTKs, Pkn1, Pkn2, Pkn8 and Pkn9, while MkapB associated with Pkn8 and Pkn9, and MkapC with Pkn8. These results indicate that there are complex PSTK networks in M. xanthus that share common modulating factors.

  • An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4-activated 6-Phosphofructokinase.
    Molecular microbiology, 2003
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary 6-Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7-fold upon phosphorylation at Thr-226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk–pkn4 deletion strain accumulated glycogen at a higher level than the wild-type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk–pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr-226-Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.

  • Activation of 6-Phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus.
    Molecular microbiology, 2002
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-Phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.

  • activation of 6 Phosphofructokinase via phosphorylation by pkn4 a protein ser thr kinase of myxococcus xanthus
    Molecular Microbiology, 2002
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-Phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.

Ryan D. Rhoades - One of the best experts on this subject based on the ideXlab platform.

  • postmortem regulation of glycolysis by 6 Phosphofructokinase in bovine m sternocephalicus pars mandibularis
    Meat Science, 2005
    Co-Authors: Ryan D. Rhoades, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Andy D King, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6-Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P<0.001) from 0.84μmol/g at d 0 to 6.54μmol/g at d 4. Fructose-6-phosphate and glucose-6-phosphate increased (P<0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33μmol/g) and was elevated to 45.9μmol/g by d 4. In the second experiment, conversion of [U-(14)C]glucose to lactate, glycogen, and CO(2) was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U-(14)C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123nmol glucose utilized per 100mg muscle per h; P=0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO(2) in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.

  • Postmortem regulation of glycolysis by 6-Phosphofructokinase in bovine M. Sternocephalicus pars mandibularis
    Meat science, 2005
    Co-Authors: Ryan D. Rhoades, D. Andy King, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6-Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P

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

  • adp dependent 6 Phosphofructokinase an extremely thermophilic non allosteric enzyme from the hyperthermophilic sulfate reducing archaeon archaeoglobus fulgidus strain 7324
    Extremophiles, 2004
    Co-Authors: Thomas Hansen, Peter Schonheit
    Abstract:

    The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. In this pathway phosphorylation of fructose-6-phosphate to fructose-1,6 bisphosphate is catalyzed by an ADP-dependent 6-Phosphofructokinase (ADP-PFK), which was purified 1,800-fold to homogeneity. The enzyme is composed of 50 kDa subunits and is eluted from gel filtration as both a homotetramer and a homodimer. It had a temperature optimum at 85°C and showed significant thermostability up to 95°C. Kinetic constants were determined for both reaction directions at pH 6.6 and 80°C. Rate dependence for all substrates followed Michaelis Menten kinetics. The apparent K m for ADP and fructose-6-phosphate (forward reaction) was 0.6 mM and 2.2 mM, respectively; the apparent V max was 1,200 U/mg. ADP-PFK catalyzed in vitro the reverse reaction, with apparent K m for fructose-1,6-bisophosphate and AMP of 5.7 and 1.4 mM, respectively, and a V max value of 85 U/mg. The enzyme did not use ATP, PPi, or acetyl phosphate as phosphoryl donor and was highly specific for fructose-6-phosphate as substrate. The A. fulgidus ADP-PFK did not phosphorylate glucose and thus differs from the bifunctional ADP-PFK/GLK from Methanococcus jannaschii. Divalent cations were required for catalytic activity; Mg2+, which was most effective, could be partially replaced by Mn2+, Ni2+, and Co2+. Enzyme activity was not allosterically regulated by classical effectors of bacterial and eukaryal ATP-PFKs, such as ADP, AMP, phosphoenolpyruvate, or citrate. N-terminal amino acid sequence showed high similarity to known ADP-PFKs. In the genome of Archaeoglobus fulgidus strain VC 16, which is closely related to strain 7324, no homologous gene for ADP-PFK could be identified.

  • ADP-dependent 6-Phosphofructokinase, an extremely thermophilic, non-allosteric enzyme from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324
    Extremophiles, 2004
    Co-Authors: Thomas Hansen, Peter Schonheit
    Abstract:

    The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. In this pathway phosphorylation of fructose-6-phosphate to fructose-1,6 bisphosphate is catalyzed by an ADP-dependent 6-Phosphofructokinase (ADP-PFK), which was purified 1,800-fold to homogeneity. The enzyme is composed of 50 kDa subunits and is eluted from gel filtration as both a homotetramer and a homodimer. It had a temperature optimum at 85°C and showed significant thermostability up to 95°C. Kinetic constants were determined for both reaction directions at pH 6.6 and 80°C. Rate dependence for all substrates followed Michaelis Menten kinetics. The apparent K _m for ADP and fructose-6-phosphate (forward reaction) was 0.6 mM and 2.2 mM, respectively; the apparent V _max was 1,200 U/mg. ADP-PFK catalyzed in vitro the reverse reaction, with apparent K _m for fructose-1,6-bisophosphate and AMP of 5.7 and 1.4 mM, respectively, and a V _max value of 85 U/mg. The enzyme did not use ATP, PPi, or acetyl phosphate as phosphoryl donor and was highly specific for fructose-6-phosphate as substrate. The A. fulgidus ADP-PFK did not phosphorylate glucose and thus differs from the bifunctional ADP-PFK/GLK from Methanococcus jannaschii . Divalent cations were required for catalytic activity; Mg^2+, which was most effective, could be partially replaced by Mn^2+, Ni^2+, and Co^2+. Enzyme activity was not allosterically regulated by classical effectors of bacterial and eukaryal ATP-PFKs, such as ADP, AMP, phosphoenolpyruvate, or citrate. N-terminal amino acid sequence showed high similarity to known ADP-PFKs. In the genome of Archaeoglobus fulgidus strain VC 16, which is closely related to strain 7324, no homologous gene for ADP-PFK could be identified.

  • ATP-dependent 6-Phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme
    Archives of microbiology, 2002
    Co-Authors: Thomas Hansen, Meike Musfeldt, Peter Schonheit
    Abstract:

    The ATP-dependent 6-Phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritima was purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 °C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant (S0.5) of 0.7 mM and a Hill coefficient of 2.2. The apparent Km for ATP was 0.2 mM and the apparent Vmax value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent Km for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent Vmax of about 13 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could partially be replaced by Mn2+ and Fe2+. Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 °C and showed a significant thermostability up to 100 °C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.

  • Sequence, expression, and characterization of the first archaeal ATP-dependent 6-Phosphofructokinase, a non-allosteric enzyme related to the Phosphofructokinase-B sugar kinase family, from the hyperthermophilic crenarchaeote Aeropyrum pernix.
    Archives of microbiology, 2001
    Co-Authors: Thomas Hansen, Peter Schonheit
    Abstract:

    The gene (ORF APF0012) encoding the ATP-dependent 6-Phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed similarity (25–40%) to members of PFK-B sugar kinases. The purified recombinant enzyme is a heterotetramer of 115 kDa, composed of 34-kDa subunits. Rate dependence (at 85 °C) on both fructose 6-phosphate (F-6-P) and ATP followed Michaelis-Menten kinetics with apparent K m values of 0.25 mM and 0.68 mM, respectively; apparent V max values were about 5 U/mg. The enzyme was specific for ATP as phosphoryl donor, but showed a broader spectrum of phosphoryl acceptors: in addition to F-6-P, glucose 6-phosphate, adenosine, fructose, ribose 5-phosphate, and ribose were accepted. Enzyme activity required divalent cations; Mg2+, which was most effective, could partially be replaced by Co2+, Ni2+, or Mn2+. The enzyme had a temperature optimum of 90 °C and showed a significant thermostability up to 100 °C. ATP-PFK activity was not allosterically regulated by classical effectors of ATP-PFKs of eukarya and bacteria, such as ADP and phosphoenolpyruvate. In accordance, this archaeal ATP-PFK did not contain the typical conserved binding sites for these effectors. This is the first report of a sequence of an archaeal ATP-PFK related to the PFK-B sugar kinase family.

  • Purification and properties of the first-identified, archaeal, ATP-dependent 6-Phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophile Desulfurococcus amylolyticus
    Archives of Microbiology, 2000
    Co-Authors: Thomas Hansen, Peter Schonheit
    Abstract:

    The ATP-dependent 6-Phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Desulfurococcus amylolyticus was purified 1500-fold to homogeneity. The enzyme had an apparent molecular mass of 140 kDa and was composed of a single type of subunit of 33 kDa suggesting a homotetrameric (α_4) structure. The N-terminal amino acid sequence did not show significant similarity to ATP-PFKs isolated from eubacteria and eukarya. Kinetic constants of the enzyme were determined for both reaction directions at pH 6 and at 85 °C. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent K _ms for ATP and fructose 6-phosphate (forward reaction) were 0.28 and 1.17 mM, respectively; the apparent V _max was about 41 U/mg. ATP could not be replaced by pyrophosphate (PP_i) or ADP as phosphoryl donor, thus defining the enzyme as an ATP-dependent PFK. In addition to ATP (100%), the enzyme accepted GTP (97%), ITP (130%), UTP (84%), CTP (55%) and, less effectively, acetyl phosphate (13%) as phosphoryl donors. Enzyme activity was not allosterically regulated by classical effectors of ATP-PFKs such as ADP, AMP, and phosphoenolpyruvate or citrate. The enzyme also catalysed in vitro the reverse reaction with an apparent K _m for fructose-1,6-bisphosphate and ADP of 16.7 and 0.5 mM, respectively, and an apparent V _max of about 4.5 U/mg. Divalent cations were required for maximal activity; Mg^2+, which was most effective, could be replaced partially by Ni^2+, Mn^2+ or Co^2+. The enzyme had a temperature optimum of 90 °C and showed a significant thermostability up to 100 °C, which is in accordance with its physiological function under hyperthermophilic conditions. This is the first description of an ATP-dependent PFK from the domain of archaea, characterized as an extremely thermophilic, non-allosteric enzyme.