22] Enzymatic formation of thiamine pyrophosphate in plants

38] Assay of thiamine pyrophosphokinase (ATP: Thiamine pyrophosphotransferase, EC 2.7.6.2) using14C- or35S-labeled thiamine

Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.

The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.

A Versatile Conformational Switch Regulates Reactivity in Human Branched-Chain α-Ketoacid Dehydrogenase

Regional Renal Adenosine Triphosphate Metabolism in Thiamine Deficiency

Pyruvate decarboxylase III: Specificity restrictions for thiamine pyrophosphate in the protein association step, sub-unit structure

Chronic alcoholism results in thiamine deficiency as a consequence of inadequate dietary intake and of impaired absorption of the vitamin. In addition, there is evidence to suggest that alcohol reduces thiamine phosphorylation to thiamine pyrophosphate (TPP) in brain. TPP is a cofactor for the pyruvate dehydrogenase complex (PDHC), alpha-ketoglutarate dehydrogenase (alpha KGDH) and transketolase (TK), three enzymes involved in cerebral glucose and energy metabolism. Pyrithiamine-induced thiamine deficiency in the rat results in early, selective, reversible reductions of alpha KGDH in brain; PDHC activities are unaffected. Reductions of alpha KGDH are accompanied by decreased aspartate, glutamate and GABA and by concomitantly increased alanine in the brain of thiamine-deficient animals. It is suggested that decreased alpha KGDH, rather than decreased PDHC constitutes 'the biochemical lesion' in thiamine deficiency encephalopathy first enunciated by Peters in the 1930s. If sufficiently prolonged and severe, thiamine deficiency results in brain cell death. Possible mechanisms involved include compromised cerebral energy metabolism and focal accumulation of lactate, both of which could result from decreased activities of alpha KGDH. In addition, it is proposed that brain cell death in severe thiamine deficiency may result from excessive release of excitotoxic amino acids. Comparable mechanisms could be involved in the cell death and in the pathogenesis of the thiamine-unresponsive symptoms of the Wernicke-Korsakoff Syndrome in humans.

Riboswitches are structured mRNA elements involved in gene regulation that respond to the intracellular concentration of specific small molecules. Binding of their cognate ligand is thought to elicit a global conformational change of the riboswitch, in addition to modulating the fine structure of the binding site. X-ray crystallography has produced detailed descriptions of the three-dimensional structures of the ligand-bound conformations of several riboswitches. We have employed small-angle X-ray scattering (SAXS) to generate low-resolution reconstructions of the ligand-free states of the ligand-binding domains of riboswitches that respond to thiamine pyrophosphate (TPP), and cyclic diguanylate (c-di-GMP), a bacterial second messenger. Comparison of the SAXS reconstructions with the crystal structures of these two riboswitches demonstrates that the RNAs undergo dramatic ligand-induced global conformational changes. However, this is not an universal feature of riboswitches. SAXS analysis of the solution behavior of several other riboswitch ligand-binding domains demonstrates a broad spectrum of conformational switching behaviors, ranging from the unambiguous switching of the TPP and c-di-GMP riboswitches to complete lack of switching for the flavin mononucleotide (FMN) riboswitch. Moreover, the switching behavior varies between examples of the same riboswitch from different organisms. The range of observed behaviors suggests that in response to the evolutionary need for precise genetic regulation, riboswitches may be tuned to function more as dimmers or rheostats than binary on/off switches.

The elongation of the third, fourth and fifth leaves of rice plants was inhibited when 30ppb or more of imazosulfuron was contained in nutrient solution when the plants were treated at the two-leaf stage, whereas root dry weight was reduced at concentrations of 1ppb and above. Addition of the branched-amino acids, valine, isoleucine and leucine at a concentration of 100ppm each alleviated the growth inhibition of the third leaf of rice plants induced by 30ppb of imazosulfuron to almost the same degree as the elongation of the control. Imazosulfuron inhibited acetolactate synthase (ALS) activity from pea seedlings (Pisum sativum) in the noncompetitive with respect to pyruvate and uncompetitive with respect to thiamine pyrophosphate (TPP) and showed the I50 at 24nM. Inhibition of ALS activity by the continuous presence of imazosulfuron was time dependent and biphasic. The constant of the initial inhibition by the herbicide was 20-fold larger than that of the final steady-state. The ALS activity from rice plants which were tolerant to imazosulfuron was sensitive to the herbicide irrespective of age or chloroplast development of the plants (I50=14 to 45nM). The results suggest that imazosulfuron acts on the plant by blocking the biosynthesis of valine, isoleucine and leucine, which is due to the direct inhibition of ALS. The herbicide is a slow-binding inhibitor of ALS activity. The binding site of imazosulfuron on the enzyme is judged not to overlap with that of pyruvate and TPP. Tolerance of rice plants to imazosulfuron does not depend on the sensitivity of ALS activity irrespective of the difference in plant age or growth condition.

Availability of abundant thiamine determines efficiency of thermogenic activation in human neck area derived adipocytes

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions.

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.

The Crystal Structure of Yeast Thiamin Pyrophosphokinase

The binding of thiamine pyrophosphate (TPP) to several enzymes has been determined by measuring cofactor-dependent activity after passage of each enzyme through a column of Sephadex G-25 to remove non-protein-bound cofactors. TPP was found to be bound irreversibly to yeast and Zymomonas pyruvate decarboxylases, Aerobacter α-acetolactate synthetase, and Escherichia glyoxylate carboligase. Cofactors were lost when Proteus pyruvate oxidase, Escherichia pyruvate dehydrogenase, and Micrococcus diacetyl carboligase were gel-filtered; the binding of TPP was strongest for diacetyl carboligase. A procedure has been devised for efficient resolution of enzymes for TPP and divalent cations. Resolved enzymes reconstituted for cofactors had properties similar to those of native enzymes. Resolved pyruvate decarboxylases from both yeast and Zymomonas mobilis failed to bind Mg++ in the absence of TPP. Cofactor reconstitution for yeast pyruvate decarboxylase was shown to be a slow process for low concentrations of TPP. TPP alone, in high concentrations, was able to activate and partially reconstitute TPP enzymes in the absence of added divalent cations. Zymomonas pyruvate decarboxylase, Aerobacter α-acetolactate synthetase, and Escherichia glyoxylate carboligase appear to be heterogeneous, in that part of each enzyme can bind TPP irreversibly; this cofactor dissociates reversibly for the remainder of the enzyme. When yeast pyruvate decarboxylase, saturated for cofactors, was gel-filtered at pH 8.0, 50% of the enzyme-bound TPP dissociated from the enzyme, the remainder being irreversibly bound. Thiazole pyrophosphate, a potent inhibitor of resolved yeast pyruvate decarboxylase, acts by binding to coenzyme sites on the enzyme. Unlike TPP, thiazole pyrophosphate was shown to be bound reversibly to the enzyme and could be displaced by high concentrations of TPP. The results obtained question the validity of calculating dissociation constants for cofactors which do not dissociate from their enzymes.