MDRZYMES · Multidrug resistance gene regulators as scaffolds for the design and evolution of artificial metalloenzymes

Multidrug resistance gene regulators as scaffolds for the design and evolution of artificial metalloenzymes

Enzymes are remarkable catalysts and the prospects of harnessing their catalytic power in industrial settings have fueled efforts to tailor biocatalysts for synthetic purposes. Indeed, mimicking the Darwinian algorithm in the laboratory, often referred to as directed evolution, has allowed for rapidly boosting the performance of existing enzymes. But what about transformations that are desirable for synthesis, yet for which no enzymes exist in nature? Creating such designer enzymes is a formidable challenge and of particular interest to chemists and biologists alike. For the former, made-to-order biocatalysts could offer a ‘green’ alternative to existing synthetic routes while for the latter creating and improving enzymes with new-to-nature activities could provide the unique opportunity to identify enzyme optimization strategies not yet explored by nature. For the creation of designer enzymes, multidrug resistance gene regulators (MDRs) are an intriguing class of proteins. In nature, these dimeric proteins serve as binding hubs for a diverse set of adversary molecules, such as antibiotics or polyaromatic compounds. Taking advantage of their remarkably promiscuous binding abilities, the Roelfes group were the first to highlight the potential of MDRs for enzyme design. Specifically, they demonstrated that the binding of planar, aromatic transition metal complexes in these binding pockets resulted in the creation of efficient hybrid enzymes for a number of abiological reactions. In an effort to expand the catalytic repertoire and improve these novel reactivities by directed evolution techniques, we incorporated unnatural amino acids featuring uniquely reactive functional groups in the hydrophobic pore of LmrR, an MDR found in Lactococcus lactis. By genetically incorporating an aniline side chain, we identified a designer enzyme, LmrR_V15pAF, that was able to catalyze a model hydrazone formation with rate accelerations orders of magnitude higher than aniline in solution. In another line of research, we evaluated the performance of various palladium complexes for cross coupling reactions inside the binding pockets of MDRs, yet failed to identify catalytically active hybrid catalysts.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз