Rational engineering of binding pocket's structure and dynamics in penicillin G acylase for selective degradation of bacterial signaling molecules

Abstract

The rapid rise of antibiotic-resistant bacteria necessitates the search for alternative, unconventional solutions, such as targeting bacterial communication. Signal disruption can be achieved by enzymatic degradation of signaling compounds, reducing the expression of genes responsible for virulence, biofilm formation, and drug resistance while evading common resistance mechanisms. Therefore, enzymes with such activity have considerable potential as antimicrobial agents for medicine, industry, and other areas of life. Here, we designed molecular gates that control the binding site of penicillin G acylase to shift its preference from native substrate to signaling molecules. Using an ensemble-based design, three variants carrying triple-point mutations were proposed and experimentally characterized. Integrated inference from biochemical and computational analyses demonstrated that these three variants had markedly reduced activity towards penicillin and each preferred specific signal molecules of different pathogenic bacteria, exhibiting up to three orders of magnitude shifts in substrate specificity. Curiously, while we could consistently expand the pockets in these mutants, the reactive binding of larger substrates was limited, either by overpromoting or overstabilizing the pocket dynamics. Overall, we demonstrated the designability of this acylase for signal disruption and provided insights into the role of appropriately modulated pocket dynamics for such a function. The improved mutants, the knowledge gained, and the computational workflow developed to prioritize large datasets of promising variants may provide a suitable toolbox for future exploration and design of enzymes tailored to disrupt specific signaling pathways as viable antimicrobial agents.