Design and Evolution of Enzymes with Non-Canonical Amino Acids

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Enzymes are exceptionally powerful catalysts that recognize molecular substrates and process them in active sites. They are generally built from just 20 amino acids, and their catalytic machinery is typically assembled from chemical groups in the amino-acid side chains. But fewer than half of these side chains contain functional groups that can participate in enzyme catalytic cycles, which severely restricts the range of mechanisms conceivable within enzyme active sites. This raises the intriguing question of whether the catalytic repertoire of enzymes could be expanded by using an extended 'alphabet' of amino acids that offers a wider range of side chains for catalysis. In recent years our group have begun to take major strides towards achieving this ambitious vision (e.g. Nature 2019, 570, 219, ACS Catalysis, 2020, 10, 2735, J. Am. Chem. Soc. 2018, 140, 1535, J. Am. Chem. Soc. 2016, 138, 11344).Our approach exploits engineered cellular translation components to selectively install non-canonical amino acids containing functional side chains. Genetically encoding the non-canonical functionality offers enormous advantages over alternative methods for chemically modifying protein structure: it greatly facilitates the production of well-defined, homogeneous proteins; it allows the non-canonical amino acid to be introduced at any site, in any protein scaffold; and, perhaps most significantly, it allows for rapid optimization of enzyme properties using directed evolution. Inspired by mechanistic strategies from small molecule organocatalysis, we have recently employed a combination of genetic code expansion, computational enzyme design and laboratory evolution to create enzymes that exploit non-canonical amino acids as catalytic nucleophiles (Nature 2019, 570, 219). This study now opens up new and exciting opportunities to enzyme designers and engineers which will be fully explored within this PhD studentship. Free from the constraints of the genetic code, the student will employ our advanced enzyme engineering techniques to create enzymes with functions not observed in Nature, that were previously thought inaccessible to the field of biocatalysis. The project will specifically aim to create enzymes that contain a functional N-heterocyclic carbene (NHC) motif embedded within the designed active site. In recent years, NHCs have emerged as powerful and highly versatile functional groups in synthetic chemistry, both as organic catalysts and as a coordinating ligands to transition metal (e.g. ruthenium, gold, palladium) catalysts including 2nd generation metathesis catalysts (Nature 2014, 510, 485). The development of general strategies for incorporating NHCs into protein active sites will therefore offer great opportunities to create highly efficient and selective catalysts for wealth of important chemical transformations. To address this objective, we will exploit engineered cellular translation components available in our laboratory to embed NHCs as cofactors into designed active sites, and subsequently explore the applications of these cofactors as organic catalysts and as ligands for gold and ruthenium mediated processes. Here we can take advantage of molecular recognition elements provided by the protein scaffold to achieve enantioselective conversions, to enhance catalytic efficiencies and to tune the electronic and structural properties of NHC cofactor. Significantly, promising starting designs can be substantially improved through iterative rounds of directed evolution to afford highly efficient and selective de novo enzymes for the production of high value molecules. The project takes a truly innovative approach to merge the fields of biocatalysis, organocatalysis and transition metal catalysis, and thus is perfectly aligned to the strategic priorities of the iCAT network.