Novel Enzymes for Biologics with Hydrolytic Activity Against Thiolactones: Computational, Catalytic and Antimicrobial Study

Background: Various thiolactones are known as biologically active compounds, capable of stimulating the development of several human diseases and quorum sensing of Gram–positive bacteria. The enzymatic hydrolysis of thiolactones represents a promising approach to preventing their action. Methods: Thirteen enzymes, including various lactonases and serine hydrolases were studied in this work using several substrates including the homocysteine thiolactone (HTL), and its derivatives the N–acetylhomocysteine thiolactone (C2–HTL) and the isobutyryl–homocystein thiolactone (i–but–HTL). The potential interactions of the ligands with the surface of enzymes molecules were predicted in silico using computational modeling and checked in wet experiments in vitro. Results: Based on the data obtained several enzymes were selected with localization of the thiolactones near their active sites, indicating the possibility of effective catalysis. The lactonase (AiiA), metallo-β-lactamase (NDM-1) and the organophosphate hydrolase with hexahistidine tag (His6–OPH) were among them. Determination of catalytic characteristics of enzymes in the hydrolytic reactions with the HTL and the C2–HTL revealed the maximal value of catalytic efficiency constant for the NDM-1 in the hydrolysis of the HTL (826 M−1 s−1). The maximal activity in the hydrolysis of C2–HTL was established for AiiA (137 M−1 s−1). The polyaspartic (PLD50) and the polyglutamic (PLE50) acids were used to obtain polyelectrolyte complexes with enzymes. The further combination of these complexes with the clotrimazole and polymyxin B possessing antimicrobial properties resulted in notable improvement of their action in relation to Staphylococcus cells. Conclusions: It was revealed that the antimicrobial activity of the polymyxin B is enhanced by 9–10 times against bacteria and yeast when combined with the His6–OPH polyelectrolyte complexes. The antimicrobial activity of clotrimazole was increased by ~7 times against Candida tropicalis cells in the case of the AiiA/PLE50/Clotrimazole combination. These results make the obtained biology attractive and promising for their further advancement to practical application.