Periodic Reporting for period 4 - enzC-Hem (Creating Versatile Metallo-Enzyme Environments for Selective C-H Activation Chemistry: Lignocellulose Deconstruction and Beyond)
Reporting period: 2022-07-01 to 2023-12-31
Enzymes are exceptionally powerful catalysts that recognize molecular substrates and process them in active sites. These biological catalysts 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 catalytic functions conceivable within enzyme active sites and limits our understanding of how enzymes operate. This project aims to overcome this fundamental limitation, by developing new biological tools that allow non-standard functional amino acids to be incorporated into enzyme active sites with high efficiency. These tools will be integrated with state-of-the-art methods in enzyme engineering and computational design to provide a truly versatile platform for building enzymes with new and improved catalytic properties, and providing us with new approaches to understand how enzymes function at the molecular level. Particular attention will be paid to the development of enzymes that accelerate challenging and valuable chemical transformations beyond those found in nature. The successful realization of these disruptive enzyme engineering strategies will ultimately lead to highly efficient and renewable catalytic systems, and thus contribute to the development of a green, efficient and sustainable chemical industry.
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.).
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.).
During this project, our lab has pioneered methods to build enzymes from an expanded alphabet of amino acids. Free from the constraints of the genetic code, entirely new modes of catalysis can now be programmed into enzymes to achieve chemistries that are far beyond those found in Nature. Our contributions to the field were recently captured in our review article 'The Road to Fully programmable Protein Catalysis' published in the leading journal Nature. Our research has also been featured in prominent magazines and scientific journals including The New Statesman, C&EN, Chemistry World and Nature, and has led to prestigious awards including the 2020 RSC Harrison Meldola Memorial Prize and the 2024 UK Blavatnik Laureate for Chemistry.