Due to the increasing resistance of microorganisms against antibiotics, many research groups are trying to identify novel biological macromolecules and metabolic pathways that could serve as targets for newly discovered antibiotics.
Two of the cellular processes, that proceed differently in prokaryotes than in eukaryotes, are amino acid metabolism and cellular communication. The protein L-threonine dehydrogenase is involved in both; it starts the conversion of the amino acid L-threonine to L-glycine, but L-threonine may also lead to the synthesis of autoinducer-3 analogues, important for quorum sensing. Unlike L-threonine dehydrogenase, succinate dehydrogenase is present in E. coli and humans, so its inhibition might also affect the host cell. We have previously shown that a pyrazole derivative 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrazole-5-ol inhibits the growth of bacterium Escherichia coli and binds to the proteins L-threonine dehydrogenase and succinate dehydrogenase. The enzyme L-threonine dehydrogenase has no human counterpart and is involved in amino acid metabolism and cellular communication. Both cellular processes operate differently in prokaryotes than in eukaryotes. L-threonine dehydrogenase initiates the conversion of the amino acid L-threonine to L-glycine or its conversion to autoinducer-3 analogues, which are important for quorum sensing. In contrast, succinate dehydrogenase, an enzyme of the citric acid cycle. Is found in E. coli and in humans, so its inhibition could also affect the host cell.
In this master’s’ thesis, our aim was to better characterize interactions between the pyrazole derivative and the two dehydrogenases. To this end, we overexpressed and purified recombinant L-threonine dehydrogenase and soluble subunits A and B of succinate dehydrogenase in E. coli. Our primary goal was to determine whether the compound had an inhibitory effect on L-threonine dehydrogenase. Inhibition of L-threonine dehydrogenase in the presence of the pyrazole derivative was monitored by measuring the fluorescence of NADH formed during the reaction. Binding of the compound to L-threonine dehydrogenase and to subunits A and B of succinate dehydrogenase was detected by isothermal titration calorimetry (ITC).
We found that the pyrazole derivative weakly binds both domains of succinate dehydrogenase and L-threonine dehydrogenase. It also inhibits the activity of L-threonine dehydrogenase, especially when the substrate concentration was lower than Km. This suggests a linear competitive mechanism of inhibition.
According to our results, the pyrazole derivative we used does not act as an antibiotic compound. Due to its binding to succinate dehydrogenase, it may have a cytotoxic effect on the host cell. The compound also likely acts as a metal ion chelator, meaning that it could potentially inhibit many other enzymes in bacterial and host cells. Nevertheless, by developing novel analogues of the compound, it may be possible to discover molecules with a narrower range of bacterial protein targets and stronger inhibition of L-threonine dehydrogenase.
|
