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The presence of an oxygen-dependent free radical is demonstrated in the thiamine diphosphate-dependent Escherichia coli 2-oxoglutarate dehydrogenase, which is a key component of the tricarboxylic acid (Krebs) cycle. The off-pathway reaction has accounted for metabolic dysfunction associated with many neurodegenerative diseases.

The analysis of orbital contributions to canonical molecular orbitals (CMOs) of six abbreviated azolium analogs of the carbanion was carried out to understand the unique function of sulfur by applying natural bond orbital (NBO) theory. The analysis has suggested the operation of an H-bonding scheme that leads to the formation of a cryptic C2 carbanion that is not accumulated and the shielding of the carbanion by the N4'-H hydrogen bond that is weakened by N1'-H deprotonation.

The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy. The g-values and (super 14)N/(super 15)N-hyperfine splittings obtained from the spectra were consistent with a planar, hyroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms.

Molecular clips functionalized by phosphate groups bind thiamine diphosphate (TPP) and S-adenosylmethionine (SAM) with high affinity in water. Various analytical tools are discussed which point toward a simultaneous insertion of both heterocyclic rings into the electron rich cavity and pave way for enzyme models and direct interference with enzymatic processes.

An experiment about molecular dynamic simulations of pyruvte decarboxylase was performed to investigate mechanism related to binding of the cofactor, thiamin diphosphate (ThDP) and the substrate in active site. It showed that a proton relay from His113 via Asp27 and Tyr290 to the carbonyl oxygen atom of the substrate might be involved in the mechanism.

Proton-nitrogen correlated NMR studies are performed on thiamin diphosphate (ThDp), which has been specifically labeled with (super 15)N at the 4'-amino group. The tremendous rate of acceleration of C2-H deprotonation in ThDp enzymes results due to the enforced conformation of the cofactor in the active site being perfectly suited to allowing intramolecular acid-base catalysis.

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on E1subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). It is evident that both intermediates, LThDP and HEThDP, exists as their 1',4'-iminopyrimidine tautomers according to the positive Cd signal in the 300-305 nm range.

The formation of a new positive CD band centered at 305 nm when the Escherichia coli pyruvate dehydrogenase complex first E1 subunit and its variants are exposed to phosphonolactylthiamin diphosphate is discussed. Results on PDHC-E1 with PLThDp establish the presence of 1'4'-iminoThDp on ThDp enzymes.

The combination of thiamin and benzaldehyde can produce benzoin, but destroys thiamin, which is due to the cleavage of the C-N bond between heterocycles. The destruction is caused from fragmentation of the conjugate of thiamin and benzaldehyde.

The first crystal structure of a eukaryotic THI6 from Candida glabrata along with several complexes that characterize the active sites responsible for the two chemical reactions, the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate and the phosphorylation of 4-methyl-5-hydroxyethylthiazole is reported. Adenosine diphospho-5[beta]-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, which is not a substrate for THI6 and 4-methyl-5-hydroxyethylthiazole phosphate which is a substrate for TH16 indicated that an unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product.

The first mechanistic insight into 2-thiosugar production in an angucycline-type antibiotic, BE-7585A, is reported. The results indicated that the sulfur insertion mechanism in 2-thiosugar biosynthesis shares similarities with that for thiamin biosynthesis.

In vivo transport assays using radiolabeled thiamin are utilized to demonstrate the involvement of the putative thiamin transporter ThiT from Lactococcus lactis in thiamin transport. Mutational analysis of the conserved residues clustered in a tryptophan-rich stretch comprising the loop between the predicted membrane spanning segments 5 and 6 combined with bindings assays of thiamin and related compounds could be used to develop a model of the high-affinity binding site.

The article studies benzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC), which are both thiamin diphosphate-dependent enzymes. Findings revealed that changes in Thr377 and Ala460 results in a substrate spectrum, which included higher activity toward pyruvate.

The various mechanisms involved in the reaction of benzoylformate decarboxylase (BFDC) with methyl benzoylphosphonate (MBP) inhibitors are discussed. The reaction is shown to lead to the formation of a tetrahedral adduct C2[alpha]-mandelyl-thiamin diphosphate.

An optimized reconstitution of the thiamin thiazole synthase (ThiG) catalyzed reaction is described. The studies have shown that the enzymatic product is an unexpected dearomatized thiazole tautomer.

The mechanism of the enzyme benzoylformate decarboxylase (BFDC), carrying out a thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, is analyzed with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). The studies have shown that the ability to directly observe the true enamine intermediate in BFDC using benzaldehyde as a substrate and the stability of this enamine intermediate has made it s potential candidate for crystallization.

Thiamin is prepared by prokaryotes and eukaryotes such as yeast and plants and the thiazole and pyrimidine moieties are prepared in separate branches of the pathway and coupled to form thiamin phosphate. Thiamin is transported into the cell and is salvaged through many routes.

A biochemical study is carried out to examine the proteins containing both an A/T-rich interaction domain (ARID) and a high mobility group (HMG)-box domain from Arabidopsis thaliana. The results showed that both the ARID and the HMG-box domain contribute to the DNA interactions of ARID-HMG1 to give the protein characteristics of ARID and HMG-box proteins.

A late intermediate, tightly bound to the active site of the Saccharomyces cerevisiae thiazole synthase, is identified as an adenylated thiazole tautomer and its reactivity is evaluated. The identification has provided an additional molecular snapshot of the complex reaction sequence catalyzed by the eukaryotic thiazole synthase and has identified the final step of the thiamin-thiazole biosynthesis.

Several structural and biochemical studies are conducted to explain the mechanisms involved in the binding of thiamine pyrophosphate (TPP) analogues to the eukaryotic riboswitches. The oxythiamine and pyrithiamine analogues of TPP compound are shown to be extremely useful for the development of different drugs.

The biochemical pathways for the decarboxylation of pyruvate that result in electrophilic substitution at an acyl carbon are discussed. The reaction involves addition of enzyme-bound thiamin diphosphate (TDP) to carbonyl group of the substrate, which provides an intermediate whose decarboxylation leads to stabilized intermediate.

Direct spectroscopic observation of thiamin diphosphate-bound intermediates performed on the enzyme benzaldehyde lyase to understand the reversible and highly enantiospecific conversion of (R)-benzoin to benzaldehyde. The observations from circular dichroism provide insight into all thiamin related intermediates which are in a chiral environment on benzaldehyde lyase.

The structural characterization of the thiamin-binding protein TbpA from Escherichia coli is presented. The structural comparison between TbpA and thiaminase-I, a thiamin-degrading enzyme showed structural similarity between two proteins evolved from a common ancestor.

The pH dependence of tautomeric forms providing the first data for the internal thermodynamic equlibria on thiamin diphosphate enzymes are reported. The dramatic influence of the protein on the internal equilibria points to conditions under which the long-elusive ylide intermediate could be stabilized.

A study shows that decarboxylation can be catalyzed by a spectator or preassociated catalyst. This explains the additional acceleration of decarboxylation seen in enzymes that generate aldehyde precursors from derivatives of thiamine diphosphate (TDP) while avoiding rapid side reactions.

The X-ray crystal structure of pyruvamide-activated yeast decarboxylase showed a flexible loop spanning residues 290 to 304 on the beta-domain of enzyme. Site-directed mutagenesis studies revealed that residue on the loop affect the activity, with some residues reducing K(sub cat)/K(sub m) by at least 1000-fold and in the pyruvamide-activated form, the loop located on the beta domain can transfer information to the cite center thiamin diphosphate located at the interface of the alpha and gamma domains.

The [sup.15.N] chemical shifts and UV spectroscopic evidence are reported for models for the 1',4'-iminopyrimidine tautomeric form of thiamin diphosphate. Both UV spectroscopic values and the [sup.15.N] chemical shift for 1',4'-iminopyrimidine tautomer serve as useful guides to the assignment of enzyme-bound signals.

The identification and characterization of the tightly bound reaction product at the active site of the Saccharomyces cerevisiae thiazole synthase is reported. The characterization of the reaction product bound at the active site of the yeast thiazole synthase has yielded insights into the biosynthesis of the thiamin thiazole in eukaryotes.

The thiamin diphosphate and flavin adenine dinucleotide-dependent pyruvate oxidase from Lacotobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Pre-steady-state and steady-state kinetics reveal that both processes are kinetically coupled.

Evidence for active-center interactions is presented for thiamin diphosphate-dependent enzymes such as yeast pyruvate decarboxylase, benzoylformate decarboxylase, and others. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound.

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decaboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. General mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.

The synthesis of the new thiamine diphosphate derivative [SnMe2(HTDP)(H2O)]Cl.H2O, its structures in solid state and in water, and the results of the kinetic experiments are described. The resulting SnMe2-holoen-zyme retains a significant proportion of the catalytic activity of the native enzyme despite the methyl groups on the metal atom.

A report is presented on the discovery of a mechanism-based inhibitor of benzoylformate decarboxylase (BFD) that covalently modifies the enzyme via active site phosphorylation, a type of mechanism-based inactivation. The report highlights that benzoylphosphonate acts as mechanism-based inhibitor BFD, with inactivation resulting from the phosphoryl-action of Ser26.

The reactivity pattern of mandelylthiamin (MTh), a conjugate of thiamin is examined. Results discovered that certain buffers accelerate its decarboxylation, although there is no apparent way that the C-C bond breaking might be stabilized by an acid or base.