Design and Evolution of Enzymes with Non-Canonical Amino Acids

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, ACS Catalysis, 2020, 10, 2735, 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 contain a functional N-heterocyclic carbene (NHC) motif embedded within the designed active site. In recent years, NHCs have emerged as powerful and highly versatile functional groups in synthetic chemistry, both as organic catalysts and as a coordinating ligands to transition metal (e.g. ruthenium, gold, palladium) catalysts including 2nd generation metathesis catalysts (Nature 2014, 510, 485). The development of general strategies for incorporating NHCs into protein active sites will therefore offer great opportunities to create highly efficient and selective catalysts for wealth of important chemical transformations. To address this objective, we will exploit engineered cellular translation components available in our laboratory to embed NHCs as cofactors into designed active sites, and subsequently explore the applications of these cofactors as organic catalysts and as ligands for gold and ruthenium mediated processes. Here we can take advantage of molecular recognition elements provided by the protein scaffold to achieve enantioselective conversions, to enhance catalytic efficiencies and to tune the electronic and structural properties of NHC cofactor. Significantly, promising starting designs can be substantially improved through iterative rounds of directed evolution to afford highly efficient and selective de novo enzymes for the production of high value molecules.
The project takes a truly innovative approach to merge the fields of biocatalysis, organocatalysis and transition metal catalysis, and thus is perfectly aligned to the strategic priorities of the iCAT network.

Planned Impact

iCAT will work with industry partners to create an holistic approach to the training of students in biocatalysis, chemocatalysis, and their process integration. Traditional graduate training typically focuses on one aspect of catalysis and this approach can severely restrict innovation and impact. Advances in technology and fundamental reaction discovery are rendering this silo-approach obsolete, and a new training modality is needed to produce the next generation of chemists and engineers who can operate across a far broader chemical continuum. iCAT will meet this challenge with a state-of-the-art CDT, equipping the next generation of scientists and engineers with the skills needed to develop future catalytic processes and create the functional molecules of tomorrow.

The UK has one of the world's top-performing chemical industries, achieving outstanding levels of growth, exports, productivity and international investment. The UK's chemical industry is a significant provider of jobs and creator of wealth, with a turnover in excess of £50 billion and a contribution of over £15 Billion of value to the UK economy [2015 figures]. iCAT will deliver highly skilled people to lead this industry across its various sectors, achieving impact through the following actions:

1. Equip the next generation of science and engineering leaders with the interdisciplinary skills and knowledge needed to work across the bio and chemo catalytic remit and build the functional molecules we need to structure society.

2. Provide a highly skilled workforce and research base, skilled in the latest methodologies, strategies and techniques of catalysis and engineering that is crucial for the UK's Chemical Industry.

3. Build the critical mass necessary to support effective cohort-based training in a world-class research environment.

4. Develop and disseminate new catalytic technologies and processes that will be taken up by industrial and academic teams around the world.

5. Encourage Industry to promote research challenges within the CDT that are of core relevance to their business.

6. Provide cohesion in the integration of biocatalysis, engineering and chemocatalysis to create a more unified voice for strategic dialogue with industry, funders and policy makers, and more generally outreach and public engagement.

7. Draw-in and bring together Industrial partners to facilitate future Industrial collaborations.

8. Benefit Industrial scientists through interactions with the CDT (e.g. training and supervisory experience, exposure to cutting-edge synthesis and catalysis etc).

9. Link with other activities in the landscape: bringing unique expertise in catalysis to, for example, externally-funded University-led initiatives, EPRSC Grand Challenge Networks, and the National Catalysis Hub.

Publications

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Hossack E (2023) Building Enzymes through Design and Evolution in ACS Catalysis

Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/S023755/1 31/03/2019 29/09/2027
2465805 Studentship EP/S023755/1 30/09/2020 29/09/2024 Euan Hossack
 
Description Participation in the Nuffield programme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Participated in the Nuffield programme by devising a summer project for an A-level student, and co-supervising this project with another PhD student. The A-level student was trained by the award holder in the use of the Chimera freeware, and taught some of the fundamentals of structural biology. The project was remote, but the A-level student was also given a tour of the building and lab the award holder works in, giving them a valuable insight into what a career in the sciences can look like.
Year(s) Of Engagement Activity 2022
URL https://www.nuffieldresearchplacements.org/