Exometabolome analysis identifies pyruvate dehydrogenase as a target for the antibiotic triphenylbismuthdichloride in multiresistant bacterial pathogens.

The desperate need for new therapeutics against notoriously antibiotic-resistant bacteria has led to a quest for novel antibacterial target structures and compounds. Moreover, defining targets and modes of action of new antimicrobial compounds remains a major challenge with standard technologies. Here we characterize the antibacterial properties of triphenylbismuthdichloride (TPBC), which has recently been successfully used against device-associated infections. We demonstrate that TPBC has potent antimicrobial activity against many bacterial pathogens. Using an exometabolome profiling approach, a unique TPBC-mediated change in the metabolites of Staphylococcus aureus was identified, indicating that TPBC blocks bacterial pyruvate catabolism. Enzymatic studies showed that TPBC is a highly efficient, uncompetitive inhibitor of the bacterial pyruvate dehydrogenase complex. Our study demonstrates that metabolomics approaches can offer new avenues for studying the modes of action of antimicrobial compounds, and it indicates that inhibition of the bacterial pyruvate dehydrogenase complex may represent a promising strategy for combating multidrug-resistant bacteria.

Co-immobilization of different enzyme activities to non-woven polyester surfaces.

Co-immobilization was applied to combine complementary enzyme reactions. Therefore, trypsin was co-immobilized together with both, lipase and alpha-amylase, onto the surface of non-woven polyester material. The progress of the immobilization reaction was directly monitored by investigating covalent fixation of the enzymes to the polyester flees using (1)H-MAS-NMR. Co-immobilization of the different types of enzymes to the polyester support showed retained enzymatic activity. However, a competition of binding to the support was observed. Increasing amounts of one type of enzyme reduced the degree of immobilization for the other type. In order to investigate the distribution of trypsin and alpha-amylase on the polyester support, the flees was treated with a mixture of rhodamine isothiocyanate labeled with anti-trypsin antibodies and fluorescein isothiocyanate labeled with anti-alpha-amylase antibodies. Using fluorescence microscopy, the co-immobilization was analyzed by selective excitation of both chromophores at 480 and 530 nm, respectively. In addition, fluorescence spectroscopy was applied by direct labeling of trypsin and lipase prior to co-immobilization to the polyester support. A special prism of plexiglass was constructed, which fit into a 10 x 10 mm fluorescence cuvette in that way that a diagonal plane was formed within the cuvette. The non-woven support was fixed in the cuvette and fluorescence spectra were obtained to characterize the amount of different enzymes linked to the support. Using FRET it was demonstrated that a uniform distribution of the various enzyme species was achieved, where the different enzyme activities are bound on the support in close neighborhood to one another.

Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity.

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.

Solvent isotope effect on the reaction catalysed by the pyruvate dehydrogenase complex from Escherichia coli.

The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the delta pK1 and delta pK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1' nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.

Immobilization of thermolysin to polyamide nonwoven materials.

In the last few years, an increasing number of biotechnological techniques have been applied to the restoration and conservation of works of art, paintings, old maps, and papers or books. Enzymes can solve problems that give restorers difficulties, although for many applications it is not possible to use soluble enzymes; therefore, it is necessary to look for suitable carriers for immobilization. Different methods for covalent immobilization of enzymes to polyamide nonwovens were tested, using thermolysin as an example. Two distinct strategies were pursued: (1). controlled, partial hydrolysis of the polymer and subsequent binding of the enzyme to the released amino and carboxy groups; and (2). attachment of reactive groups directly to the polyamide without disintegrating the polymeric structure (O-alkylation). Different spacers were used for covalent fixation of the enzyme in both cases. The enzyme was fixed to the released amino groups by glutaraldehyde, either with or without a spacer. Either way, active enzyme could be immobilized to the matrix. However, intense treatment caused severe damage to the stability of the nonwoven fabric, and reduced the mechanical strength. Conditions were investigated to conserve the nonwoven fabric structure while obtaining near-maximum immobilized enzyme activity. Immobilization of the enzyme to the released carboxy group after acid hydrolysis was performed using dicyclohexylcarbodiimide. In comparison to the enzyme bound via the amino group, the yield of immobilized enzyme activity was slightly lower when benzidine was taken as spacer and still lower with a 1,6-hexanediamine spacer. O-alkylation performed with dimethylsulfate caused severe damage to the nonwoven fabric structure. Considerably better results were obtained with triethyloxonium tetrafluoroborate. As the spacers 1,6-hexanediamine and adipic acid dihydrazide were used, activation for immobilizing thermolysin was performed with glutaraldehyde, adipimidate, and azide. With the exception of azide, all combinations of spacers and activation reagents gave high yields of immobilized enzyme activity. Thermolysin immobilized by this technique showed a remarkably improved stability with respect to elevated temperature, extreme pH values, and reduced polarity. The nonwoven fabric can be stored for weeks without loss of enzyme activity by washing with distilled water and drying.