Enzymes with an Expanded Amino Acid Alphabet

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Combining computational design and directed evolution is a powerful approach to create enzymes for non-biological reactions. However, the range of chemistries accessible is restricted by Nature's alphabet of canonical amino acids, which contain limited functionality and are not optimal for many biocatalytic transformations. Genetic code expansion (GCE) allows the site-selective installation of 'chemically programmed' non-canonical amino acids (ncAAs) into enzyme active sites, providing opportunities to access organocatalytic mechanisms not represented in Nature. A useful example are thioureas, which are a valuable class of small-molecule organocatalysts capable of activating carbonyl compounds through dual hydrogen-bonding interactions to promote a broad range of chemical transformations.Previously in the group, pyrrolysyl-tRNA synthetase/PyltRNACUA pairs have successfully been engineered to encode several lysine-derived thioureas in response to reassigned amber stop codons (UAG). We are currently working on expanding the scope of encodable thioureas and have so far successfully incorporated one urea analogue. With these translation components in hand, we will be able to explore a range of chemical transformations within computationally designed active sites, eventually optimising these enzymes via directed evolution. This approach thus combines the efficiency and selectivity of protein catalysts, with the broad range of reactivities accessible with small molecule organocatalysts.