Antibiotic resistance has been observed since the beginning of antibiotic development. The excessive and incorrect use of antimicrobial agents, the overprescription of antibiotics, and their widespread use in industry have led to the uncontrolled spread of antibiotic resistance across microbial populations. The search for new antibiotics and their targets is of crucial importance, therefore, the identification of protein targets is one of the ways to discover and develop new therapeutics. In the case of combating pathogenic organisms, we would search for proteins that are crucial for the uninterrupted functioning of the organism and consequently find out how to disable their function. Hydroxynaphthoic acid derivatives, biologically active small molecules, have previously been shown to inhibit the growth of some gram-positive bacteria, including Staphylococcus aureus, and that they bind to WrbA. The aim of this master's thesis was to prepare NAD(P)H:quinone oxidoreductase from E. coli (WrbA) and its two homologous proteins from S. aureus (labelled ffp1 and ffp2), and to test the effect of some hydroxynaphthoic acid derivatives on the enzymatic activity of the aforementioned proteins. All three proteins were successfully expressed and isolated, incubated with the cofactor FMN, and their activity was confirmed by monitoring the oxidation of the substrate NADH to NAD+ by measuring the absorbance. We successfully tested the effect of five different hydroxynaphthoic acid derivatives on all three enzymes. The derivatives that most successfully inhibited all three enzymes were 1-hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid. In comparison to the positive control, 1-hydroxy-2-naphthoic acid inhibited the activity of WrbA by 39 %, ffp1 by 67 % and ffp2 by 82 %, while 2-hydroxy-1-naphthoic acid inhibited WrbA by 49 %, ffp1 by 86 % and ffp2 by 87 %. The enzyme that was most susceptible to inhibition by all tested derivatives was ffp2. Finally, we characterized the two unknown homologues, ffp1 and ffp2, by determining kinetic parameters and predicting their structures with the tool AlphaFold. It turned out that all three proteins have similar values of kinetic parameter, and a similar folding of their structures, so it is likely that they belong to the same protein family. 1-hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid could represent a potential new antimicrobial compound, but in the future, it would be necessary to investigate why cell growth is completely inhibited, while enzyme activity is only partially inhibited, and whether there may be other protein targets in S. aureus that could explain this.
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