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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.

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 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.

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.

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.

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.

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.

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 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.

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.