Due to its biofilm forming ability, which allows it to survive in adverse conditions, S. aureus is a major problem for antibiotic treatment. It was recently discovered that anaerobic biofilm formation requires menaquione. DHNA, which is a downstream metabolite in menaquinone biosynthesis, inhibits the second enzyme of the classical menaquinone biosnynthesis pathway MenD. In this thesis, we prepared recombinant proteins MenF and MenD, which are the first two enzymes in the classical pathway. MenD is the first committed enzyme of the pathway, which converts isochorismate to 2 succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC). Our aim was to prepare isochorismate from chorismate using the enzyme MenF and then use isochorismate to observe MenD activity and test the potential inhibitory effect of DHNA like hydroxynaphthoic acid derivatives on the MenD enzyme. In our experiments, we successfully prepared both recombinant proteins and used MenF to convert chorismate to isochorismate, but were unable to test the inhibitory effects of the derivatives on MenD because the enzyme was inactive due to its inability to tetramerise. We presume that this is due to an extended N-terminus introduced in molecular cloning. In future work, the N-terminal tag cleavage should be optimized or a C-terminal tag introduced instead. Inhibition of MenD represents a good starting point for developing new antibiotics focusing on menaquinone biosynthesis inhibition, so further research in this area would be beneficial.
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