Dietary thiamin level influences levels of its diphosphate form and thiamin-dependent enzymic activities of rat liver.
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This study was prompted by our incomplete understanding of the mechanism responsible for the clinical benefits of pharmacological doses of thiamin in some patients with maple syrup urine disease (MSUD) and the question of whether thiamin diphosphate (TDP), a potent inhibitor of the activity of the protein kinase that phosphorylates and inactivates the isolated branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex, affects the activity state of the complex. Rats were fed a chemically-defined diet containing graded levels of thiamin (0, 0.275, 0.55, 5.5, and 55 mg thiamin/kg diet). Maximal weight gain was attained over a 3-wk period only in rats fed diets with 5.5 and 55 mg thiamin/kg. Feeding rats the thiamin-free diet for just 2 d caused loss of nearly half of the TDP from liver mitochondria. Three more days caused over 70% loss, an additional 3 wk, over 90%. Starvation for 2 d had no effect, suggesting a mechanism for conservation of TDP in this nutritional state. Mitochondrial TDP was higher in rats fed pharmacological amounts of thiamin (55 mg thiamin/kg diet) than in rats fed adequate thiamin for maximal growth. Varying dietary thiamin had marked but opposite effects on the activities of alpha-ketoglutarate dehydrogenase (alpha-KGDH) and BCKDH. Thiamin deficiency decreased alpha-KGDH activity, increased BCKDH activity, and increased the proportion of BCKDH in the active, dephosphorylated, state. Excess dietary thiamin had the opposite effects. TDP appears to be more tightly associated with alpha-KGDH than BCKDH in thiamin-deficient rats, perhaps denoting retention of alpha-KGDH activity at the expense of BCKDH activity. Thus, thiamin deficiency and excess cause large changes in mitochondrial TDP levels that have a major influence on the activities of the keto acid dehydrogenase complexes. (+info)
Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase.
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Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding. (+info)
Chemical modification of lysine and arginine residues of bovine heart 2-oxoglutarate dehydrogenase: effect on the enzyme activity and regulation.
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Chemical modification of arginine and lysine residues of bovine heart 2-oxoglutarate dehydrogenase with phenylglyoxal and pyridoxal 5'-phosphate inactivated the enzyme, indicating the importance of these residues for the catalysis. Inactivation caused by pyridoxal 5'-phosphate was prevented in the presence of thiamine pyrophosphate and Mg2+ allowing the assumption that lysine residues participate in binding of the cofactor. (+info)
Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids.
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In the third step of the alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant. (+info)
Interplay of organic and biological chemistry in understanding coenzyme mechanisms: example of thiamin diphosphate-dependent decarboxylations of 2-oxo acids.
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With the publication of the three-dimensional structures of several thiamin diphosphate-dependent enzymes, the chemical mechanism of their non-oxidative and oxidative decarboxylation reactions is better understood. Chemical models for these reactions serve a useful purpose to help evaluate the additional catalytic rate acceleration provided by the protein component. The ability to generate, and spectroscopically observe, the two key zwitterionic intermediates invoked in such reactions allowed progress to be made in elucidating the rates and mechanisms of the elementary steps leading to and from these intermediates. The need remains to develop chemical models, which accurately reflect the enzyme-bound conformation of this coenzyme. (+info)
Reduced flux through the purine biosynthetic pathway results in an increased requirement for coenzyme A in thiamine synthesis in Salmonella enterica serovar typhimurium.
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Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in Salmonella enterica serovar Typhimurium. Prior work showed that panE mutants (panE encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of panE mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth. (+info)
Crystal versus solution structures of thiamine diphosphate-dependent enzymes.
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The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits. (+info)
The structural basis of substrate activation in yeast pyruvate decarboxylase. A crystallographic and kinetic study.
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The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed. (+info)