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

Periodic Reporting for period 1 - MDRZYMES (Multidrug resistance gene regulators as scaffolds for the design and evolution of artificial metalloenzymes)

Reporting period: 2017-03-01 to 2019-02-28

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.
We began our search for a designer enzyme featuring an unnatural amino acid as catalytic residue, by incorporating an aniline side chain (p-aminophenylalanine, pAF) at various positions in the hydrophobic binding pocket of LmrR. With anilines being well-known nucleophilic organocatalysts we evaluated the performance of pAF-containing LmrR variants in a model hydrazone formation reaction. Indeed, we identified one variant, LmrR_V15pAF, which displayed promising catalytic activity for this transformation.

To gain further insight into the mode of catalysis of LmrR_V15pAF we performed a detailed characterization of the designer enzyme. Trapping of an intermediate formed between one substrate and the aniline side chain attested on the unique reactivity of the introduced pAF residue; full labeling was observed for LmrR_V15pAF but neither for any of the other pAF-containing variants tested nor the wild-type protein. A detailed kinetic characterization showed that embedding the unnatural amino acid in the LmrR binding pocket increased the reactivity of this side chain by a factor >550. This rate acceleration is orders of magnitude higher than the ones that can be achieved by small molecule aniline derivatives. Two factors contribute to the proficiency of LmrR in hydrazone formation reactions. On the one hand, the promiscuous binding pocket of LmrR provides a suitable environment to recruit the reactants and increase their effective molarity. On the other hand, judiciously placing the pAF residue inside the hydrophobic pore increases the reactivity of the aniline side chain and results in a more efficient nucleophilic catalyst. Although the catalytic machinery of LmrR_V15pAF is working as designed, it is a primitive apparatus consisting of a catalytic residue placed in a hydrophobic environment. We reasoned that directed evolution techniques could be used to optimize the role of the unnatural side chain in catalysis. Toward this end, we developed a medium-throughput screening protocol that allowed us to identify point mutations that further boosted the performance of LmrR_V15pAF. Future efforts to shuffle beneficial mutations and identify synergistic combinations are likely to yield even more proficient LmrR variants.

During the period Dr. Mayer was supported by the Marie Skłodowska-Curie fellowship, results of this work have been presented at the ICMSE 2017 in Basel (poster). After the conclusion of the fellowship, the work will be presented at the NCCC XIX in in Noordwijkerhout. A manuscript describing the design and characterization of LmrR_V15pAF is scheduled for publication in early 2018, with a follow-up publication describing the directed evolution of the designer enzyme later the same year.
Creating made-to-order enzymes with the ability to catalyze abiological transformations is a formidable challenge and diverse engineering strategies have been employed to design artificial enzymes with novel activities. What these efforts have in common is that they typically rely on the canonical set of amino acids to introduce these novel functions. However, the incorporation of unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. The identified LmrR_V15pAF represents the first entry in the list of man-made biocatalysts that feature an unnatural amino acid as a catalytic residue. Moreover, the directed evolution efforts in this proposal augur well that the activity of unnatural side chains can be tailored in a similar fashion as their canonical counterparts. The future creation and optimization of novel catalysts with non-natural, catalytic side chains could provide a fascinating insight in novel optimization strategies not yet explored by natural enzymes. Ultimately, we anticipate that the developed design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
Enzyme design with unnatural amino acids as catalytic residues