During this project we have taken major strides toward achieving our ambitious vision of building enzymes from an expanded amino acid alphabet. Our approach exploits engineered cellular translation components to selectively install non-canonical amino acids containing functional side chains. We have exploited this approach to create several metalloenzymes with non-canonical coordination environments (J. Am. Chem. Soc. 2018, 140, 1535. Chem. Eur. J. 2018, 24, 11821). A non-canonical ligand has also allowed us to unravel the active site features that control the reactivity of iconic ferryl intermediates in haem and non-heme iron enzymes (ACS Catalysis, 2020, 10, 2735. JACS Au, 2021, 1, 913. ACS Catalysis, 2024, 14, 15, 11584–11590.) These studies have recently been extended to create a series of de novo heme enzymes with both natural and artificial cofactors for performing challenging oxidations and carbene transfer processes (J. Am. Chem. Soc. 2023, 145, 26, 14307–14315). In parallel, we have been able to capture & characterize elusive reactive intermediates in copper dependent lytic polysaccharide monooxygenases, a recently discovered family of enzymes that show great potential as auxiliary biocatalysts for commercially viable cellulose deconstruction. These studies reveal an in-built enzyme repair mechanism involving the His-brace active site that protects the enzymes from oxidative damage during catalytic turnover (J. Am. Chem. Soc. 2023, 145, 20672-20682).
We have also recently demonstrated that our protein engineering strategies can be extended beyond metalloenzyme design. Taking inspiration from the field of small molecule organocatalysis, we have used 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. Nature Chemistry 2021, 14, 313. Curr. Opin. Chem. Biol. 2020, 55, 136. Nat Commun. 2024, 15, 1956. Nature Catalysis, 2022, 5, 673). We have also shown that our approaches can be used to develop enantioselective photoenzymes that operate via triplet energy transfer mechanisms, a powerful mode of reactivity in organic synthesis that is currently beyond the reach of biocatalysis (Nature, 2022, 611, 709–714.).
