The Experts below are selected from a list of 1242 Experts worldwide ranked by ideXlab platform
Georg A Sprenger - One of the best experts on this subject based on the ideXlab platform.
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novel mode of inhibition by d tagatose 6 phosphate through a heyns rearrangement in the active site of Transaldolase b variants
Acta Crystallographica Section D-biological Crystallography, 2016Co-Authors: L Stellmacher, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Sarah Schneider, Anne K. SamlandAbstract:Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type Transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 A and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 A. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.
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acid base catalyst discriminates between a fructose 6 phosphate aldolase and a Transaldolase
Chemcatchem, 2015Co-Authors: L Stellmacher, Tatjana Sandalova, Sebastian Leptihn, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:The residues responsible for binding the catalytic water molecule were interchanged between the closely related enzymes fructose 6-phosphate aldolase A (FSAA) and Transaldolase B (TalB) from Escherichia coli. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6-phosphate aldolase and Transaldolase activity, stability, pH dependence of activity, pKa value of the essential lysine residue and their three dimensional structure. The double variant TalBE96Q F178Y showed improved aldolase activity with an apparent kcat of 4.3 s−1. The experimentally determined pKa values of the catalytic lysine residue revealed considerable differences: In FSAA, this lysine residue is deprotonated at assay conditions (pKa 5.5) whereas it is protonated in TalB (pKa 9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid.
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the Transaldolase family new synthetic opportunities from an ancient enzyme scaffold
ChemInform, 2011Co-Authors: Anne K. Samland, Georg A Sprenger, Madhura Rale, Wolfdieter FessnerAbstract:Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalytic carboligation by aldolases offers a green, uniquely regio- and stereoselective tool with which to perform these transformations. Recent advances in the field, fueled by both discovery and protein engineering, have greatly improved the synthetic opportunities for the atom-economic asymmetric synthesis of chiral molecules with potential pharmaceutical relevance. New aldolases derived from the Transaldolase scaffold (based on Transaldolase B and fructose-6-phosphate aldolase from Escherichia coli) have been shown to be unusually flexible in their substrate scope; this makes them particularly valuable for addressing an expanded molecular range of complex polyfunctional targets. Extensive knowledge arising from structural and molecular biochemical studies makes it possible to address the remaining limitations of the methodology by engineering tailored biocatalysts.
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Transaldolase from biochemistry to human disease
The International Journal of Biochemistry & Cell Biology, 2009Co-Authors: Anne K. Samland, Georg A SprengerAbstract:The role of the enzyme Transaldolase (TAL) in central metabolism, its biochemical properties, structure, and role in human disease is reviewed. The nearly ubiquitous enzyme Transaldolase is a part of the pentose phosphate pathway and transfers a dihydroxyacetone group from donor compounds (fructose 6-phosphate or sedoheptulose 7-phosphate) to aldehyde acceptor compounds. The phylogeny of Transaldolases shows that five subfamilies can be distinguished, three of them with proven TAL enzyme activity, one with unclear function, and the fifth subfamily comprises Transaldolase-related enzymes, the recently discovered fructose 6-phosphate aldolases. The three-dimensional structure of a bacterial (Escherichia coli TAL B) and the human enzyme (TALDO1) has been solved. Based on the 3D-structure and mutagenesis studies, the reaction mechanism was deduced. The cofactor-less enzyme proceeds with a Schiff base intermediate (bound dihydroxyacetone). While a Transaldolase deficiency is well tolerated in many microorganisms, it leads to severe symptoms in homozygous TAL-deficient human patients. The involvement of TAL in oxidative stress and apoptosis, in multiple sclerosis, and in cancer is discussed.
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replacement of a phenylalanine by a tyrosine in the active site confers fructose 6 phosphate aldolase activity to the Transaldolase of escherichia coli and human origin
Journal of Biological Chemistry, 2008Co-Authors: Sarah Schneider, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:Abstract Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of Transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human Transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
Gunter Schneider - One of the best experts on this subject based on the ideXlab platform.
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novel mode of inhibition by d tagatose 6 phosphate through a heyns rearrangement in the active site of Transaldolase b variants
Acta Crystallographica Section D-biological Crystallography, 2016Co-Authors: L Stellmacher, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Sarah Schneider, Anne K. SamlandAbstract:Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type Transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 A and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 A. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.
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acid base catalyst discriminates between a fructose 6 phosphate aldolase and a Transaldolase
Chemcatchem, 2015Co-Authors: L Stellmacher, Tatjana Sandalova, Sebastian Leptihn, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:The residues responsible for binding the catalytic water molecule were interchanged between the closely related enzymes fructose 6-phosphate aldolase A (FSAA) and Transaldolase B (TalB) from Escherichia coli. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6-phosphate aldolase and Transaldolase activity, stability, pH dependence of activity, pKa value of the essential lysine residue and their three dimensional structure. The double variant TalBE96Q F178Y showed improved aldolase activity with an apparent kcat of 4.3 s−1. The experimentally determined pKa values of the catalytic lysine residue revealed considerable differences: In FSAA, this lysine residue is deprotonated at assay conditions (pKa 5.5) whereas it is protonated in TalB (pKa 9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid.
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replacement of a phenylalanine by a tyrosine in the active site confers fructose 6 phosphate aldolase activity to the Transaldolase of escherichia coli and human origin
Journal of Biological Chemistry, 2008Co-Authors: Sarah Schneider, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:Abstract Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of Transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human Transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
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Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the Transaldolase of Escherichia coli and human origin.
The Journal of biological chemistry, 2008Co-Authors: Sarah Schneider, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of Transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human Transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
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crystal structure of decameric fructose 6 phosphate aldolase from escherichia coli reveals inter subunit helix swapping as a structural basis for assembly differences in the Transaldolase family
Journal of Molecular Biology, 2002Co-Authors: Stina Thorell, Melanie Schürmann, Georg A Sprenger, Gunter SchneiderAbstract:Abstract Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R -factor of 19.9% ( R free 21.3%). The subunit folds into an α/β barrel, with the catalytic lysine residue on barrel strand β4. It is very similar in overall structure to that of bacterial and mammalian Transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical Transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of Transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the Transaldolase family.
Anne K. Samland - One of the best experts on this subject based on the ideXlab platform.
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novel mode of inhibition by d tagatose 6 phosphate through a heyns rearrangement in the active site of Transaldolase b variants
Acta Crystallographica Section D-biological Crystallography, 2016Co-Authors: L Stellmacher, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Sarah Schneider, Anne K. SamlandAbstract:Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type Transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 A and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 A. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.
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acid base catalyst discriminates between a fructose 6 phosphate aldolase and a Transaldolase
Chemcatchem, 2015Co-Authors: L Stellmacher, Tatjana Sandalova, Sebastian Leptihn, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:The residues responsible for binding the catalytic water molecule were interchanged between the closely related enzymes fructose 6-phosphate aldolase A (FSAA) and Transaldolase B (TalB) from Escherichia coli. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6-phosphate aldolase and Transaldolase activity, stability, pH dependence of activity, pKa value of the essential lysine residue and their three dimensional structure. The double variant TalBE96Q F178Y showed improved aldolase activity with an apparent kcat of 4.3 s−1. The experimentally determined pKa values of the catalytic lysine residue revealed considerable differences: In FSAA, this lysine residue is deprotonated at assay conditions (pKa 5.5) whereas it is protonated in TalB (pKa 9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid.
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the Transaldolase family new synthetic opportunities from an ancient enzyme scaffold
ChemInform, 2011Co-Authors: Anne K. Samland, Georg A Sprenger, Madhura Rale, Wolfdieter FessnerAbstract:Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalytic carboligation by aldolases offers a green, uniquely regio- and stereoselective tool with which to perform these transformations. Recent advances in the field, fueled by both discovery and protein engineering, have greatly improved the synthetic opportunities for the atom-economic asymmetric synthesis of chiral molecules with potential pharmaceutical relevance. New aldolases derived from the Transaldolase scaffold (based on Transaldolase B and fructose-6-phosphate aldolase from Escherichia coli) have been shown to be unusually flexible in their substrate scope; this makes them particularly valuable for addressing an expanded molecular range of complex polyfunctional targets. Extensive knowledge arising from structural and molecular biochemical studies makes it possible to address the remaining limitations of the methodology by engineering tailored biocatalysts.
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Transaldolase from biochemistry to human disease
The International Journal of Biochemistry & Cell Biology, 2009Co-Authors: Anne K. Samland, Georg A SprengerAbstract:The role of the enzyme Transaldolase (TAL) in central metabolism, its biochemical properties, structure, and role in human disease is reviewed. The nearly ubiquitous enzyme Transaldolase is a part of the pentose phosphate pathway and transfers a dihydroxyacetone group from donor compounds (fructose 6-phosphate or sedoheptulose 7-phosphate) to aldehyde acceptor compounds. The phylogeny of Transaldolases shows that five subfamilies can be distinguished, three of them with proven TAL enzyme activity, one with unclear function, and the fifth subfamily comprises Transaldolase-related enzymes, the recently discovered fructose 6-phosphate aldolases. The three-dimensional structure of a bacterial (Escherichia coli TAL B) and the human enzyme (TALDO1) has been solved. Based on the 3D-structure and mutagenesis studies, the reaction mechanism was deduced. The cofactor-less enzyme proceeds with a Schiff base intermediate (bound dihydroxyacetone). While a Transaldolase deficiency is well tolerated in many microorganisms, it leads to severe symptoms in homozygous TAL-deficient human patients. The involvement of TAL in oxidative stress and apoptosis, in multiple sclerosis, and in cancer is discussed.
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replacement of a phenylalanine by a tyrosine in the active site confers fructose 6 phosphate aldolase activity to the Transaldolase of escherichia coli and human origin
Journal of Biological Chemistry, 2008Co-Authors: Sarah Schneider, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:Abstract Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of Transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human Transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
András Perl - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial dysfunction in the liver and antiphospholipid antibody production precede disease onset and respond to rapamycin in lupus prone mice
Arthritis & Rheumatism, 2016Co-Authors: Zachary Oaks, Katalin Banki, Thomas Winans, Tiffany Caza, David Fernandez, Yuxin Liu, Steve K Landas, András PerlAbstract:Objective Antiphospholipid antibodies (aPL) constitute a diagnostic criterion of systemic lupus erythematosus (SLE), and aPL have been functionally linked to liver disease in patients with SLE. Since the mechanistic target of rapamycin (mTOR) is a regulator of oxidative stress, a pathophysiologic process that contributes to the development of aPL, this study was undertaken in a mouse model of SLE to examine the involvement of liver mitochondria in lupus pathogenesis. Methods Mitochondria were isolated from lupus-prone MRL/lpr, C57BL/6.lpr, and MRL mice, age-matched autoimmunity-resistant C57BL/6 mice as negative controls, and Transaldolase-deficient mice, a strain that exhibits oxidative stress in the liver. Electron transport chain (ETC) activity was assessed using measurements of oxygen consumption. ETC proteins, which are regulators of mitochondrial homeostasis, and the mTOR complexes mTORC1 and mTORC2 were examined by Western blotting. Anticardiolipin (aCL) and anti–β2-glycoprotein I (anti-β2GPI) autoantibodies were measured by enzyme-linked immunosorbent assay in mice treated with rapamycin or mice treated with a solvent control. Results Mitochondrial oxygen consumption was increased in the livers of 4-week-old, disease-free MRL/lpr mice relative to age-matched controls. Levels of the mitophagy initiator dynamin-related protein 1 (Drp1) were depleted while the activity of mTORC1 was increased in MRL/lpr mice. In turn, mTORC2 activity was decreased in MRL and MRL/lpr mice. In addition, levels of aCL and anti-β2GPI were elevated preceding the development of nephritis in 4-week-old MRL, C57BL/6.lpr, and MRL/lpr mice. Transaldolase-deficient mice showed increased oxygen consumption, depletion of Drp1, activation of mTORC1, and elevated expression of NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), a pro-oxidant subunit of ETC complex I, as well as increased production of aCL and anti-β2GPI autoantibodies. Treatment with rapamycin selectively blocked mTORC1 activation, NDUFS3 expression, and aPL production both in Transaldolase-deficient mice and in lupus-prone mice. Conclusion In lupus-prone mice, mTORC1-dependent mitochondrial dysfunction contributes to the generation of aPL, suggesting that such mechanisms may represent a treatment target in patients with SLE.
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the pathogenesis of Transaldolase deficiency
Iubmb Life, 2007Co-Authors: András PerlAbstract:The signaling networks that mediate cell growth, differentiation, and survival are dependent on complex metabolic and redox pathways. Metabolism of glucose through the pentose phosphate pathway (PPP) fulfills two unique functions: formation of ribose 5-phosphate for the synthesis of nucleotides, RNA, and DNA in support cell growth and formation of NADPH for biosynthetic reactions and neutralization of reactive oxygen intermediates (ROI). Balancing of NADPH and ROI levels by the PPP enzyme Transaldolase (TAL) regulates the mitochondrial trans-membrane potential (Δψm), a critical checkpoint of ATP synthesis and cell survival. While complete deficiency of glucose 6-phosphate dehydrogenase (G6PD) or transketolase (TK) is lethal, TAL-deficient mice developed normally with the exception of male sterility due to structural and functional damage of sperm cell mitochondria. Recently, two cases of complete TAL deficiency have been reported in patients with liver cirrhosis which results from increased cell death of hepatocytes. Delineation of the cell type-specific role that TAL plays in the PPP and cell death signal processing will be critical for understanding the pathogenesis of TAL deficiency. iubmb Life, 59: 1-9, 2007
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deletion of ser 171 causes inactivation proteasome mediated degradation and complete deficiency of human Transaldolase
Biochemical Journal, 2004Co-Authors: Craig E Grossman, Katalin Banki, Cornelis Jakobs, Sandor Vajda, Nanda M Verhoeven, Brian Niland, Christina Stancato, Marjo S Van Der Knaap, Lawrence Brown, András PerlAbstract:Homozygous deletion of three nucleotides coding for Ser-171 (S171) of TAL-H (human Transaldolase) has been identified in a female patient with liver cirrhosis. Accumulation of sedoheptulose 7-phosphate raised the possibility of TAL (Transaldolase) deficiency in this patient. In the present study, we show that the mutant TAL-H gene was effectively transcribed into mRNA, whereas no expression of the TALΔS171 protein or enzyme activity was detected in TALΔS171 fibroblasts or lymphoblasts. Unlike wild-type TAL-H–GST fusion protein (where GST stands for glutathione S-transferase), TALΔS171–GST was solubilized only in the presence of detergents, suggesting that deletion of Ser-171 caused conformational changes. Recombinant TALΔS171 had no enzymic activity. TALΔS171 was effectively translated in vitro using rabbit reticulocyte lysates, indicating that the absence of TAL-H protein in TALΔS171 fibroblasts and lymphoblasts may be attributed primarily to rapid degradation. Treatment with cell-permeable proteasome inhibitors led to the accumulation of TALΔS171 in whole cell lysates and cytosolic extracts of patient lymphoblasts, suggesting that deletion of Ser-171 led to rapid degradation by the proteasome. Although the TALΔS171 protein became readily detectable in proteasome inhibitor-treated cells, it displayed no appreciable enzymic activity. The results suggest that deletion of Ser-171 leads to inactivation and proteasome-mediated degradation of TAL-H. Since TAL-H is a regulator of apoptosis signal processing, complete deficiency of TAL-H may be relevant for the pathogenesis of liver cirrhosis.
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znf143 mediates basal and tissue specific expression of human Transaldolase
Journal of Biological Chemistry, 2004Co-Authors: Craig E Grossman, Katalin Banki, Yueming Qian, András PerlAbstract:Transaldolase regulates redox-dependent apoptosis through controlling NADPH and ribose 5-phosphate production via the pentose phosphate pathway. The minimal promoter sufficient to drive chloramphenicol acetyltransferase reporter gene activity was mapped to nucleotides -49 to -1 relative to the transcription start site of the human Transaldolase gene. DNase I footprinting with nuclear extracts of Transaldolase-expressing cell lines unveiled protection of nucleotides -29 to -16. Electrophoretic mobility shift assays identified a single dominant DNA-protein complex that was abolished by consensus sequence for transcription factor ZNF143/76 or mutation of the ZNF76/143 motif within the Transaldolase promoter. Mutation of an AP-2alpha recognition sequence, partially overlapping the ZNF143 motif, increased TAL-H promoter activity in HeLa cells, without significant impact on HepG2 cells, which do not express AP-2alpha. Cooperativity of ZNF143 with AP-2alpha was supported by supershift analysis of HeLa cells where AP-2 may act as cell type-specific repressor of TAL promoter activity. However, overexpression of full-length ZNF143, ZNF76, or dominant-negative DNA-binding domain of ZNF143 enhanced, maintained, or abolished Transaldolase promoter activity, respectively, in HepG2 and HeLa cells, suggesting that ZNF143 initiates transcription from the Transaldolase core promoter. ZNF143 overexpression also increased Transaldolase enzyme activity. ZNF143 and Transaldolase expression correlated in 21 different human tissues and were coordinately upregulated 14- and 34-fold, respectively, in lactating mammary glands compared with nonlactating ones. Chromatin immunoprecipitation studies confirm that ZNF143/73 associates with the Transaldolase promoter in vivo. Thus, ZNF143 plays a key role in basal and tissue-specific expression of Transaldolase and regulation of the metabolic network controlling cell survival and differentiation.
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Relationship between posttranslational modification of Transaldolase and catalase deficiency in UV-sensitive repair-deficient xeroderma pigmentosum fibroblasts and SV40-transformed human cells.
Free radical biology & medicine, 2001Co-Authors: Fabienne Lachaise, András Perl, Monique Vuillaume, Ghislaine Martin, Christianne Drougard, Maurice Wegnez, Alain Sarasin, Leela Daya-grosjeanAbstract:Xeroderma Pigmentosum (XP) is a rare recessively inherited human disease associated with a hypersensitivity to ultraviolet radiation. The ultraviolet component of sunlight can initiate and promote the formation of cutaneous tumors as seen in the skin cancer-prone XP patients. Previously, we have found that the low activity of the NADPH-dependent antioxydant enzyme, catalase, which we have observed in XP diploid fibroblasts and SV40-tranformed cells, could be restored by the addition of NADPH. Here we have analyzed Transaldolase, which regulates NADPH levels produced by the pentose phosphate pathway in order to examine how it influences the catalase activity regulated in XP and SV40-transformed cells. We find that Transaldolase activity is high in XP and SV40-transformed human fibroblasts, whereas Transaldolase transcription is unchanged, suggesting that modification of Transaldolase activity is due to a posttranslational modification of the protein. Two-dimensional electrophoresis analysis has allowed us to identify a complex set of Transaldolase isoforms and to postulate that the phosphorylation of specific isoforms could be correlated with the different enzymatic activities seen. Our results show that high Transaldolase activity corresponds to a low catalase activity in SV40-transformed cells and in fibroblasts from XP patients who have a high predisposition to develop skin cancer.
Tatjana Sandalova - One of the best experts on this subject based on the ideXlab platform.
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novel mode of inhibition by d tagatose 6 phosphate through a heyns rearrangement in the active site of Transaldolase b variants
Acta Crystallographica Section D-biological Crystallography, 2016Co-Authors: L Stellmacher, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Sarah Schneider, Anne K. SamlandAbstract:Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type Transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 A and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 A. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.
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acid base catalyst discriminates between a fructose 6 phosphate aldolase and a Transaldolase
Chemcatchem, 2015Co-Authors: L Stellmacher, Tatjana Sandalova, Sebastian Leptihn, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:The residues responsible for binding the catalytic water molecule were interchanged between the closely related enzymes fructose 6-phosphate aldolase A (FSAA) and Transaldolase B (TalB) from Escherichia coli. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6-phosphate aldolase and Transaldolase activity, stability, pH dependence of activity, pKa value of the essential lysine residue and their three dimensional structure. The double variant TalBE96Q F178Y showed improved aldolase activity with an apparent kcat of 4.3 s−1. The experimentally determined pKa values of the catalytic lysine residue revealed considerable differences: In FSAA, this lysine residue is deprotonated at assay conditions (pKa 5.5) whereas it is protonated in TalB (pKa 9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid.
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replacement of a phenylalanine by a tyrosine in the active site confers fructose 6 phosphate aldolase activity to the Transaldolase of escherichia coli and human origin
Journal of Biological Chemistry, 2008Co-Authors: Sarah Schneider, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:Abstract Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of Transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human Transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
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Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the Transaldolase of Escherichia coli and human origin.
The Journal of biological chemistry, 2008Co-Authors: Sarah Schneider, Tatjana Sandalova, Gunter Schneider, Georg A Sprenger, Anne K. SamlandAbstract:Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of Transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human Transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
