Design and Evolution of Photo-Enzymes for Stereoselective Transformations of Nitrogen Radicals

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

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, 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 promote enantioselective photo-redox transformations. In recent years, the Leonori group (project partner) have developed a wealth of valuable photo-redox processes involving the intermediate generation of nitrogen radicals. At present, these transformations produce racemic products, and the development of enantioselective versions of these reactions remains a key unresolved challenge. To address the objective, we will exploit engineered cellular translation components available in our laboratory to embed organic cofactors with suitable properties for mediating photo-induced electron transfers into designed active sites. Here we can take advantage of molecular recognition elements provided by the protein scaffold to achieve enantioselective conversions. Significantly, promising starting designs can be substantially improved through iterative rounds of directed evolution to afford highly efficient and selective photo-redox enzymes for the production of high value molecules.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/S023755/1 01/04/2019 30/09/2027
2279462 Studentship EP/S023755/1 01/10/2019 30/09/2023 Jonathan Trimble