Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Inhibition by fructose 1,6-bisphosphate of transaldolase from Escherichia coli.

The effect of fructose 1,6-bisphosphate (Fru 1,6-P2) on the regulatory enzymes of pentose phosphate pathway of Escherichia coli was examined. Fru 1,6-P2 inhibited E. coli transaldolase (EC 2.2.1.2) competitively against fructose 6-phosphate and uncompetitively against erythrose 4-phosphate, whereas Fru 1,6-P2 did not affect glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Kinetic results can be explained by assuming that transaldolase has two kinds of binding sites for Fru 1,6-P2: a competitive binding site for fructose 6-phosphate and a second binding site on the enzyme-erythrose 4-phosphate complex. Fru 1,6-P2 increased resulting from the stimulation of glycolysis, can inhibit transaldolase and further participates in the elevation of the concentration of ribose 5-phosphate that can be preferentially utilized for anabolic reaction in exponential phase of E. coli.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

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 Å 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 ß-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 Å. 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.

Arabinose 5-phosphate covalently inhibits transaldolase.

Arabinose 5-phosphate (A5P) is the aldopentose version of the ketohexose fructose 6-phosphate (F6P), having identical stereochemistry but lacking atoms corresponding to the 1-carbon and 1-hydroxyl. Despite structural similarity and conservation of the reactive portion of F6P, F6P acts as a substrate whereas A5P is reported to be an inhibitor of transaldolase. To address the lack of A5P reactivity we determined a crystal structure of the Francisella tularensis transaldolase in complex with A5P. This structure reveals that like F6P, A5P forms a covalent Schiff base with active site Lys135. Unlike F6P, A5P binding fails to displace an ordered active site water molecule. Retaining this water necessitates conformational changes at the A5P-protein linkage that possibly hinder reactivity. The findings presented here show the basis of A5P inhibition and suggest an unusual mechanism of competitive, reversible-covalent transaldolase regulation.

Elevation of mitochondrial transmembrane potential and reactive oxygen intermediate levels are early events and occur independently from activation of caspases in Fas signaling.

Stimulation of the CD95/Fas/Apo-1 receptor leads to apoptosis through activation of the caspase family of cysteine proteases and disruption of the mitochondrial transmembrane potential (Deltapsim). We show that, in Jurkat human T cells and peripheral blood lymphocytes, Fas-induced apoptosis is preceded by 1) an increase in reactive oxygen intermediates (ROI) and 2) an elevation of Deltapsim. These events are followed by externalization of phosphatidylserine (PS), disruption of Deltapsim, and cell death. The caspase inhibitor peptides, DEVD-CHO, Z-VAD.fmk, and Boc-Asp.fmk, blocked Fas-induced PS externalization, disruption of Deltapsim, and cell death, suggesting that these events are sequelae of caspase activation. By contrast, in the presence of caspase inhibitors, ROI levels and Deltapsim of Fas-stimulated cells remained elevated. Because ROI levels and Deltapsim are regulated by the supply of reducing equivalents from the pentose phosphate pathway (PPP), we studied the impact of transaldolase (TAL), a key enzyme of the PPP, on Fas signaling. Overexpression of TAL accelerated Fas-induced mitochondrial ROI production, Deltapsim elevation, activation of caspase-8 and caspase-3, proteolysis of poly(A)DP-ribose polymerase, and PS externalization. Additionally, suppression of TAL diminished these activities. Therefore, by controlling the balance between mitochondrial ROI production and metabolic supply of reducing equivalents through the PPP, TAL regulates susceptibility to Fas-induced apoptosis. Early increases in ROI levels and Deltapsim as well as the dominant effect of TAL expression on activation of caspase-8/Fas-associated death domain-like IL-1beta-converting enzyme, the most upstream member of the caspase cascade, suggest a pivotal role for redox signaling at the initiation of Fas-mediated apoptosis.

Inhibition of the catalytic activity of human transaldolase by antibodies and site-directed mutagenesis.

Transaldolase is a key enzyme of the pentose phosphate pathway. While antibody (Ab) 169, directed against the N-terminal 139 residues of human transaldolase (TAL-H), had no effect on enzyme activity, Ab 12484 raised against full length and functional recombinant TAL-H inhibited catalytic activity. This tentatively mapped the catalytic site to the C-terminal 140-336 amino acid portion of TAL-H. Dihydroxyacetone transfer reactions catalyzed by transaldolase depend on Schiff base formation by a lysine residue. Replacement of lysine-142 by glutamine using site-directed mutagenesis resulted in a complete loss of enzyme activity, suggesting that lysine-142 is essential for the catalytic activity of TAL-H.

[Standards of hourly excretion of B group vitamins with urine for children aged 9 to 13 years]. / Normy chasovoi ékskretsii s mochoi vitaminov gruppy V dlia detei 9-13 let.

Values of hour excretion of vitamins B1, B2, B6 and PP with urine in children of 9-13 years, studied under conditions of normal consumption of these vitamins, were estimated considering the correlation between the vitamins B concentration in blood and excretion of their metabolites with urine as well as using these parameters dependence on content of the vitamins in daily ration; for this purpose 35 adult persons and 31 children of both sexes were examined. Normal rate of riboflavin excretion with urine constituted 10-11 micrograms/h in children of this age, while of thiamine-11-12 micrograms/h. Under conditions of normal thiamine consumption, activity of erythrocyte transketolase, measured after preinactivation of transaldolase, exceeded 35 mumol/h/I ml of erythrocytes. Rates of excretion with urine of 4-pyridoxic acid and I-methyl nicotinamide were similar both in children and in adult persons and were equal to more than 40 micrograms/h and 400-600 micrograms/h, respectively.

[A comparative assessment of the biochemical criteria of provision of the body with thiamine]. / Sravnitel'naia otsenka biokhimicheskikh kriteriev obespechennosti organizma tiaminom.

Activity of transketolase in erythrocyte hemolysates, estimated by means of measurement of sedoheptulose-7-phosphate content without preinactivation of transaldolase, proved to be 3-4-fold lower as compared with the values observed after preinactivation of transaldolase. The values of thiamine diphosphate (TDP) effect did not depend on conditions of the transketolase reaction, as well as on the colorimetric procedure used for determination of sedoheptulose-7-phosphate. A high inverse correlation was found between the value of TDP effect and an excretion of thiamine with urine in adult patients and in children of 9-13 years old. Normal excretion of thiamine with urine constituted 11-12 micrograms/h in the children of this age as judged from the TDP effect-thiamine excretion in children of this age.

Specific irreversible inhibition of enzymes concomitant to the oxidation of carbanionic enzyme-substrate intermediates by hexacyanoferrate (III).

Four different enzymes, class I fructose-1,6-biphosphate aldolase from rabbit muscle, class II fructose-1,6-bisphosphate aldolase from yeast, transaldolase, and transketolase, are inactivated progressively in the presence of their specific substrates and hexacyanoferrate(III). The inactivation is strictly linked to the oxidation of the carbanionic enzyme-substrate intermediates of these enzymes reported previously [Healy, M. J. and Christen, P. (1973) Biochemistry, 12, 35]. However, the loss of activity is not due to the products of this oxidation, i.e. to hexacyanoferrate(II), or to the oxidation product of the substrate such as hydroxypyruvaldehyde phosphate in the case of aldolase [Healy, M. J. and Christen, P. (1972) J. Am. Chem. Soc. 94, 7911]. The inactivation is not reversed on removal of low-molecular-weight compounds by gel filtration or extensive dialysis indicating a covalent modification of the enzyme. The rate of inactivation obeys saturation kinetics with respect to substrate concentration. Hence, the modifying agent is a transiently reactive intermediate formed during the oxidation of the carbanionic enzyme-substrate intermediate by hexacyanoferrate(III).