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A photoreactive acetogenin is synthesized. The results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit is challenged.

The new dimers of avenanthramide B are synthesized by a reaction of peroxidase and avenanthramide B in the presence of hydrogen peroxide. It is shown that all the compounds are classified into lignanamides that are formed by an 8'-8' coupling reaction between two avenanthramide B units, which are found in oat leaves.

Trianglelac-acetogenins, a new class of inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) act differently from ordinary inhibitors such as rotenone and piericidin A. The results show that binding site of trianglelac-acetogenins is not identical to the binding site of ubiquinone and downstream of that of ordinary inhibitors.

Studies were conducted on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase, which are useful for explaining the structural and functional feature of the terminal electron transfer step of enzyme. It suggested that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion.

Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring are synthesized and it is suggested that their inhibition mechanism may be different from that of common acetogenins. The results obtained demonstrate that deltalac-acetogenins are a novel type of inhibitor at the terminal electron-transfer step pf bovine complex I.

We developed a computational technique to assist in the large-scale identification of charged metabolites. The electrophoretic mobility of metabolites in capillary electrophoresis-mass spectrometry (CE-MS) was predicted from their structure, using an ensemble of artificial neural networks (ANNs). Comparison between relative migration times of 241 various cations measured by CE-MS and predicted by a trained ANN ensemble produced a correlation coefficient of 0.931. When we used our technique to characterize all metabolites listed in the KEGG ligand database, the correct compounds among the top three candidates were predicted in 78.0% of cases. We suggest that this approach can be used for the prediction of the migration time of any cation and that it represents a powerful method for the identification of uncharacterized CE-MS peaks in metabolome analysis.

A novel two-dimensional separation method, which hyphenated chromatography and electrophoresis, was developed for analysis of Bacillus subtilis metabolites. Micro-liquid chromatography (LC) with a monolithic silica-ODS column was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Concentration strategies, namely, dynamic pH junction and sweeping, were selectively employed to interface the two dimensions, which proved to be beneficial for the detection of metabolites. For system evaluation, an artificial sample containing 54 standard metabolites was separated according to their hydrophobicity by micro-LC with gradient mode. The early-eluting fractions were separated by capillary zone electrophoresis in combination with dynamic pH junction, while the late-eluting fractions were separated by sweeping micellar electrokinetic chromatography. The middle fractions were analyzed by both modes of CE. Under the optimum conditions, all the components in the artificial sample could be well resolved. The method was applied to profile B. subtilis metabolites. Some crucial metabolites were identified. This method provided great potential for resolving complex biological samples containing compounds having different characteristics.

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamide-adenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 [micro]mol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 [mu]mol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.

A loopless mutant, with its loop replaced by three residues of glycine, is synthesized to study the function of the loop in Escherichia coli. The loopless mutant enzyme is similar to the wild type enzyme in the crystal structure. The loop replacement enhances the kinetic values of the glycine, which is involved in the loop replacement.

Adenosine(5')polyphospho(5')pyridoxals (APn-PLs; n = 2, 3, 4) were examined for affinity labeling of glutathione synthetase from Escherichia coli B. It was found that AP4-PL has the highest affinity and labels Lys-18 specifically. X-ray diffraction analysis of AP4-PL-soaked crystal has revealed binding of its adenosine moiety in the active-site crevice where the adenosine moiety of ATP binds. Thus, the adenosine moiety of AP4-PL binds at the nucleotide-binding site of the enzyme.

Anion-exchange resin-catalyzed transhydrocyanation and enzymatic acetylation allow the highly stereoselective conversion of aldehydes into optically active cyanohydrin acetates. When racemic cyanohydrins undergo acylation with isopropenyl acetate,the Pseudomonas cepacia lipase allows only the (S)-isomer to react while the (R)-isomer is regressively racemized via transhydrocyanation. This eventually leads to 63-100% conversion in up to 94% enantiomeric excess. The conversion technique may be used in preparing (S)-cyanohydrins from commercially available(R)-isomers.

The flexible loop of glutathione synthetase in Escherichia coli B was studied through limited proteolysis and kinetic measurements for the wild type and mutant enzymes. Protein fragmentation and enzyme inactivation was seen as the peptide bond on position arginine 233 and glycine 234of the enzyme was disrupted. Stability of the loop is provided by the presence of substrates in the presence of proteases which is more evident in the wild-type enzyme although glycine did not show any protective effects. The findings suggest the role of the loop as binding site for substrate and its protective function for enzyme stability and activity.