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Creating Versatile Metallo-Enzyme Environments for Selective C-H Activation Chemistry: Lignocellulose Deconstruction and Beyond

Periodic Reporting for period 2 - enzC-Hem (Creating Versatile Metallo-Enzyme Environments for Selective C-H Activation Chemistry: Lignocellulose Deconstruction and Beyond)

Reporting period: 2019-07-01 to 2020-12-31

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 such as selective functionalizations of inert C-H bonds. 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.
In the first 2.5 years of this project we have taken major strides toward achieving our ambitious research vision. We have established engineered cellular translation components to selectively install non-canonical amino acids containing new functional side chains, and are making progress in developing mutually orthogonal translation systems to allow selective introduction of two distinct non-standard amino acids into the same active site (unpublished data). Genetically encoding the non-canonical functionality 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, we have shown that this approach allows rapid optimization of enzyme properties using directed evolution. We have exploited these advanced protein engineering methods to create several metalloenzymes with non-canonical coordination environments (e.g. 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 (metal-oxo) intermediates in haem enzymes (ACS Catalysis, 2020, 10, 2735). In parallel, we have been able to capture 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 (manuscript under review). 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, our lab 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. Curr. Opin. Chem. Biol. 2020, 55, 136).
In the first 2.5 years of this project, we have shown how incorporating new functional amino acids into established enzyme design and evolution workflows provides new tools to understand how enzymes function at the molecular level, and offers a truly versatile platform for creating enzymes with augmented properties and new functions. These studies have opened up exciting new avenues in protein catalysis which will be fully explored within the remainder of the research program. Key avenues of ongoing and future investigation include: the development of enzymes containing multiple functional non-canonical amino acids; a detailed understanding of the factors controlling the structures and reactivities of high valent metal-oxo intermediates in copper and iron dependent enzymes; the development of enzymes capable of promoting non-biological functionalizations of unactivated C-H bonds, including azidations, C-C and C-N formations; the use of ultra-high throughput screening platforms (>10^7 variants per day) to rapidly engineer enzymes with an expanded amino acid alphabet, and the development of de novo metalloproteins capable of supporting catalysis by biological (e.g. Fe and Cu) and non-biological cofactors. The realization of these objectives will allow the field of enzyme design and biocatalysis to move beyond the state-of-the-art, where we rely on a handful of functional amino acid building blocks, to a scenario where diverse functional groups and engineering strategies are available to produce de novo enzymes with interesting catalytic properties.
New Tools for Enzyme Design and Engineering
Reengineering Metalloenzymes with Non-Canonical Ligands