Exploiting the distinctive catalysis of chemically modified enzymes

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre


Enzymes are the catalysts that nature uses to accelerate chemical reactions. Because enzymes are spectacularly efficient catalysts that operate in water at room temperature, they have tremendous potential to be exploited in the 'green' manufacture of chemicals such as drug molecules. However, a major limitation for application in chemical synthesis is that enzymes are highly selective for a particular starting material, and it is not always possible to find an enzyme that nature has evolved that is suitable for a specific application. We propose to modify a specific enzyme such that it may be exploited in the catalysis of a much wider range of useful chemical transformations.

Enzymes are proteins that are constructed from 20 amino acid building blocks. Essentially, the sequence of amino acids in a protein is analogous to a sequence of coloured beads in a necklace. Although nature exploits some very useful amino acid building blocks, the functions of enzymes are nonetheless limited by the fact that there are only 20 amino acids that are usually used to construct proteins. Here, we propose to exploit new methods to prepare proteins with a much wider range of amino acid building blocks. We have demonstrated the ability to position these new amino acids, termed non-canonical amino acids, throughout the active site of an enzyme and that the specificity of the enzyme may be changed by using the non-canonical amino acids in ways that cannot be achieved using the 'natural' building blocks. In this proposal we will first expand the range of specificity of the enzyme by positioning the new amino acids at a range of positions and then using an established assay we will find new reactions that can occur only when the new amino acid is present.

In the second part of the work we will develop new assays to allow us to extend the possibilities even further. Firstly we will make use of nuclear magnetic resonance (NMR) to follow the reaction using labelled substrates, and then we will make use of NMR techniques from the realm of drug discovery (named fragment-based drug design) to search for a wider range of substrates used when we have non-canonical amino acids in the enzyme. We will also extend the chemical method for incorporation of the non-canonical amino acid.

Finally, and most excitingly, the ability to insert 'unnatural' amino acid building blocks also opens the way to building completely novel enzyme chemistries - possible using one of the inserted 'unnatural' amino acids, but not possible using any of the 20 natural amino acids. For this we have taken inspiration from a field of chemistry termed organocatalysis. Here relatively simple organic molecules can catalyse reactions, and the coupling of this area with the specificity and catalytic power of enzymes will be used to generate enzymes with huge potential for new, greener routes to a range of important chemicals.

The modified enzymes that we will discover will be able to catalyse reactions for which enzymes are not currently available. Such enzymes would have tremendous value in the 'green' synthesis of complex biologically active molecules such as drugs.

Technical Summary

Protein engineering and directed evolution have provided the tools to harness the phenomenal power of enzyme catalysis for useful purposes, but both methods have been limited to the replacement of amino acids with any of the 20 natural, or canonical, amino acids. However on occasions nature makes use of post-translational modifications to increase the range of activities that can be catalysed. We have chosen to use a chemical modification approach to insert non-canonical amino acids into an enzyme in order to expand the substrate scope of the enzyme, to explore the structure-activity relationships in active sites bearing non-canonical amino acids and, excitingly, to open the way to catalysis of reaction chemistries not possible using solely the canonical amino acids.

We have already demonstrated the feasibility of the approach by regenerating enzyme activity in an aldolase where the essential catalytic lysine residue is replaced by a thia-lysine residue and we have further demonstrated that substrate specificity can be switched by the inclusion of a non-canonical amino acid in the active site where none of the canonical amino acids can cause the altered specificity. Here we will exploit this ability to produce aldolases with altered specificity and stereochemistry that will expand their range to useful synthetic reactions. We will first use our existing assay but will expand the range significantly by the development of new NMR based assays; one following the reaction with 13C-labelled substrates and the second drawing on ideas from fragment-based drug design to expand the catalytic range. Finally we will use the ability to insert non-canonical amino acids to increase the chemistry possible in the enzyme active site by taking lessons from organocatalysis to use both enamine and iminium catalysis using non-canonical analogues of lysine in the active site.

Planned Impact

The beneficiaries of the research are:

1. Companies engaged in the manufacture of fine chemicals (including pharmaceuticals and agrochemicals)
Enzymes are useful as biocatalysts in many areas including the production of valuable fine chemicals for the pharmaceutical and chemical industries. The global market for industrial enzymes was nearly $4.5 billion in 2012 and is expected to reach $7.1 billion by 2018. Industrial biocatalysis is now widely viewed as the 'third wave' of biotechnology, following the pharmaceutical and agricultural waves. Enzymes are attractive to industry, not only because they are highly selective and efficient, but also because they catalyse the production of relatively pure products, minimising waste generation. These catalysts perform regiospecific, chemospecific and stereospecific reactions that are challenging for conventional chemistry, and do so under mild conditions with relatively non-toxic reagents.

Advances in protein engineering have ushered in a new era of industrial biocatalysis, resulting in both new products and in improvements in manufacturing processes. However, despite these advances, the fundamental limitations of the chemistry of the 20 naturally-occurring amino acids limit the potential reach of biocatalysts. In this grant, we will demonstrate that chemical modification - to extend the amino acid alphabet - can extend the ability of biocatalysts to meet the future needs of the high value chemicals manufacturing sector.

We will organise a 1-day meeting (in Year 2) to which we will invite end-users from a range of appropriate companies. This activity will disseminate the early results from the programme to end-users and ensure ongoing alignment with future industrial need. We will disseminate the results to researchers (including end-users) through publication in international peer-reviewed journals, if necessary after protection of underlying IP. We will also continue to work closely with the University's Press Office to issue press releases to coincide with high-impact publications; this approach has previously catalysed follow-on highlight articles in high-impact journals (eg Science, Nature series, C&E News, Cell series) and in trade magazines/websites.

The applicants will ensure that exploitable IP is appropriately protected using Leeds' robust approach. They will work as appropriate with specialised innovation managers who will assist the development of pathways to commercialisation/translation, and will coordinate with Leeds' Commercialisation Services unit and venture capital partners.

2. The general public
The ultimate beneficiaries of the research are the general public who benefit from products (including medicines) based on high value chemicals.

We will develop a short animated film for dissemination to illustrate catalysis and the value of non-canonical amino acids to alter enzyme activity. The postdoctoral co-worker will receive public communication training prior to preparation of the film. The film will be uploaded to appropriate websites (eg YouTube, Leeds' LUTube) to allow global dissemination, and will be targeted at AS/A2 pupils and science teachers through Leeds' ongoing outreach activities. We will also continue to engage with the general public eg through invited visits to schools, education conferences, and Café Scientifique (Leeds and Manchester).

In addition, we will continue to issue press releases to coincide with major publications; this has previously catalysed media coverage that engages the general public (eg Daily Telegraph, London Evening Standard, Yorkshire Evening Post, local radio).

The accompanying Pathway to impact will thus meet the following objectives:
PtI1. To disseminate the research to end-users and to align with future end-user needs;
PtI2. To engage the general public in biocatalysis; and
PtI3. To exploit the commercial value of the novel chemically-modified enzymes.


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Windle CL (2017) Extending enzyme molecular recognition with an expanded amino acid alphabet. in Proceedings of the National Academy of Sciences of the United States of America

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Windle CL (2017) Aldolase-catalysed stereoselective synthesis of fluorinated small molecules. in Current opinion in chemical biology

Description It is an exciting time for protein engineers wishing to create novel enzymes with useful properties -advances in structural methods allows better rational design; advances in directed evolution open the way to exploring wide areas of sequence space; and advances in computational approaches permits the de novo design of activities for the first time. However these methods have been restricted to engineering with the 20 canonical, ribosomally decoded amino acids. The pioneering methods for creation of new tRNA/tRNA synthetase pairsand the development of new chemical mutagenesis methods now opens the way to protein engineering with an extended amino acid alphabet.

We have used chemical modification/mutagenesis of an enzyme to introduce a range of 13 non-canonical amino acids at 12 positions throughout the enzyme active site and we have systematically screened for activity with a range of substrates. This approach has enabled the identification of an enzyme activity and an alteration of substrate specificity that, crucially, cannot be engineered by saturation mutagenesis using the 20 canonical amino acids. We have further characterised the new enzyme structurally to understand how the introduction of the non-canonical amino acid has brought about the specificity switch.

In recent work we have introduced a wide range of new side chains (57) and have screened for new activities with aromatic aldehydes thus searching for new activities for NAL. Our work has identified a number of active substrate/side chain combinations and some of these have been fully characterised kinetically and structurally.

This work is of broad interest to the protein engineering community and to those interested in structure-activity relationships in proteins as well as to the chemical biology community and those working specificially on the use of enzymes/aldolases for synthesis. Our findings to date demonstrate that the extended amino acid alphabet increases the arsenal of tools available to protein engineers and opens the way to the creation of new enzymes with chemistries beyond that which Nature has evolved.
Exploitation Route This research opens the door to engineering with non-canonical amino acids and shows what is possible.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Public engagement event at Astbury Conversation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The Astbury Conversation is a biennial event bringing an audience of several hundred to hear a scientific symposium focussed around a Nobel prize winner, with a public engagement event to publicise the work supported to an audience rangining from targetted schools, through general school children to university students. The next Astbury Conversation will be in 202 and a link is provided below
Year(s) Of Engagement Activity 2018
URL https://astburyconversation.leeds.ac.uk/ehome/index.php?eventid=200183132&
Description Research seminar and mentoring talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talk to a University Biology Club to introduce the new topic area, to provoke thinking and discussion and encourage development of research careers.
Year(s) Of Engagement Activity 2017