Imine Reductases: Biochemistry, Engineering and Application
Lead Research Organisation:
University of York
Department Name: Chemistry
Abstract
The synthesis of biologically active pharmaceutical and agrochemical compounds requires that chemical intermediates are synthesised in a very specific way, giving only one of two possible isomeric forms, as the unwanted form may be inactive or even toxic. Some of the most important chemical intermediates in pharmaceuticals synthesis are amines, nitrogen containing compounds that form part of well-known drugs such as beta-blockers and painkillers such as ibuprofen. It is a significant challenge to synthesise amines in single isomer form, and increasingly both academics and industrialists are turning to enzymes - nature's biological catalysts - as an efficient way of making these compounds. Not only do enzymes naturally possess the required selectivity to make single isomers, they are also attractive from the perspective of sustainable, green chemistry, as they work at ambient temperature and pressure, do not require toxic chemical reagents to work, and generate lower hazard waste than some non-biological catalysts. Many kinds of enzyme have been used to make single-isomer amines, but a new class of enzymes 'imine reductases (IREDs)' has recently been unearthed, which offers new and improved ways to synthesise these important compounds. One of the major advantages relates to reaction yield; while many of the competing enzymatic routes result in only 50% maximal theoretical yield, IREDs can deliver 100% yield of the single isomer form required. In this project we propose to take our early work on IREDs and to develop this with a view to offering valuable new enzyme catalysts for industry. We will first engineer commonly used industrial bacteria to make large amounts of the IRED enzymes, and then we will thoroughly test their ability to catalyse the industrial reactions of interest. We will use X-ray crystallography to determine the molecular structure of these enzymes, and this will allow us to make conclusions about how they work at a molecular level. If we learn more about how the enzymes work, we can then use protein engineering techniques to make the enzymes work better on the reactions of interest, but also expand their applicability so that they work on new reactions that are not catalysed by the natural enzymes. We can also use the structural information to improve the way that the enzymes will perform under industrial process conditions. In the end, a comprehensive review of this class of IREDs will be performed, resulting in important new information on a largely unexplored group of enzymes, and valuable catalysts for the production of important industrial synthetic intermediates.
Technical Summary
Efficient methods for the asymmetric synthesis of chiral amines are important for the pharmaceutical industry as amines constitute prestige targets in pharmaceutical chemistry. Increasingly, industry is turning to biocatalytic routes for the production of chiral amines as enzymes not only offer superior selectivity in the production of single enantiomer products, but comply with many of the precepts of green and sustainable chemistry. Many different kinds of enzymes have been applied to the enantioselective synthesis of amines, including hydrolases, amine oxidases and transaminases, but these can suffer from limited yields, poor stability and reaction equilibrium issues. One under-explored area of biocatalysis for the production of chiral amines is the use of putative 'imine reductases' (IREDs). The transformation of prochiral imines to chiral amines using IREDs would offer quantitative asymmetric route to amine products of high optical purity. In this project, we will build on preliminary investigations to conduct a comprehensive study into the biochemistry and application of NAD(P)H-dependent IREDs. Target genes will be cloned, expressed and the IREDs applied to the synthesis of a wide range of amine products in both single-enzyme reactions and as part of cascades with other amine-transforming enzymes such as transaminases and amine oxidases. The structures of interesting IREDs will be pursued using X-ray crystallography, and structural information used to guide mutational investigations into mechanism and specificity in these enzymes. Structures will also be used to guide targeted combinatorial mutagenesis experiments that look to alter substrate range and improve activity of IREDs. In vitro evolution techniques based on random mutagenesis, in combination with relevant screens, will also be used to improve catalytic characteristics and process suitability.
Planned Impact
The development of selective, efficient and green methods of chemicals synthesis has wide-ranging consequences that go far beyond the academic sphere. The pharmaceutical chemicals industry is one of the major beneficiaries of research into these improved synthetic methods, which delivers technological and economically beneficial solutions to the problems of synthesising chiral intermediates in an efficient and environmentally benign manner. The investigation and development of such techniques is increasingly important as the proportion of IB processes within industrial chemistry is thought to increase to 20% by 2015 (Source: Horizon 2020 EU Framework Programme). The PI and CoPI collaborate extensively with representatives of major UK and other international phamaceutical companies (GSK, Dr Reddy's, Merck, Codexis et al.). Some of these activities are enabled through the Centre of Excellence for Biocatalysis, Biotransformations and Biomanufacture (CoEBio3) of which the Co-PI is director. The contacts within CoEBio3 will allow communication of results, when appropriate, through face-to-face meetings and presentations to both individual companies and meetings of the combined industrial affiliates of CoEBio3. Industrial representatives are often laboratory scientists in biotransformation laboratories who will be able to quickly transfer information on new techniques and developments to their own groups within companies, within a 2-4 y timescale, dependent on the publication of results. Given the importance of the pharmaceutical sector to the UK, improved processes for the chemical industry will make a clear contribution to economic competitiveness.
Outside the academic and industrial parties that most directly gain advantage from new technology advances within the project, relevant UK government stakeholders also derive benefit in general from successful developments in Industrial Biotechnology, as these help to publicise the benefits of investing in IB and also inform the development of future policy. Organisations such as the BiS-funded Bioscience Knowledge Transfer Network are well placed to use these case studies to champion IB in the presence of companies who had not previously considered using this technology. The general benefits of IB such as that used in the project can be communicated through Bioscience KTN activites such as visits, newsletters, webinars and workshops.
Last, the wider beneficiaries of the work will be the general public. As consumers of pharmaceutical products, the public benefits from the more efficient production of these compounds, and also from the development of new pharmaceuticals that can be accessed using new technology. More generally and in the longer term, the incorporation of industrial biotechnology techniques into pharmaceutical manufacture allows the public to benefit from the improved lifestyles and environment associated with a more efficient, sustainable and environmentally benign chemicals industry. The impact of these developments on the public can be communicated at local and international level using press releases, articles in popular science journals, and at public engagement events such as the York Festival of Ideas and the Manchester Science Festival.
Outside the academic and industrial parties that most directly gain advantage from new technology advances within the project, relevant UK government stakeholders also derive benefit in general from successful developments in Industrial Biotechnology, as these help to publicise the benefits of investing in IB and also inform the development of future policy. Organisations such as the BiS-funded Bioscience Knowledge Transfer Network are well placed to use these case studies to champion IB in the presence of companies who had not previously considered using this technology. The general benefits of IB such as that used in the project can be communicated through Bioscience KTN activites such as visits, newsletters, webinars and workshops.
Last, the wider beneficiaries of the work will be the general public. As consumers of pharmaceutical products, the public benefits from the more efficient production of these compounds, and also from the development of new pharmaceuticals that can be accessed using new technology. More generally and in the longer term, the incorporation of industrial biotechnology techniques into pharmaceutical manufacture allows the public to benefit from the improved lifestyles and environment associated with a more efficient, sustainable and environmentally benign chemicals industry. The impact of these developments on the public can be communicated at local and international level using press releases, articles in popular science journals, and at public engagement events such as the York Festival of Ideas and the Manchester Science Festival.
Organisations
Publications
Aleku G
(2018)
Kinetic Resolution and Deracemization of Racemic Amines Using a Reductive Aminase
in ChemCatChem
Aleku G
(2016)
Stereoselectivity and Structural Characterization of an Imine Reductase (IRED) from Amycolatopsis orientalis
in ACS Catalysis
Aleku GA
(2017)
A reductive aminase from Aspergillus oryzae.
in Nature chemistry
Cosgrove SC
(2018)
Imine Reductases, Reductive Aminases, and Amine Oxidases for the Synthesis of Chiral Amines: Discovery, Characterization, and Synthetic Applications.
in Methods in enzymology
Eger E
(2020)
Correction to "Inverted Binding of Non-natural Substrates in Strictosidine Synthase Leads to a Switch of Stereochemical Outcome in Enzyme-Catalyzed Pictet-Spengler Reactions".
in Journal of the American Chemical Society
Eger E
(2020)
Inverted Binding of Non-natural Substrates in Strictosidine Synthase Leads to a Switch of Stereochemical Outcome in Enzyme-Catalyzed Pictet-Spengler Reactions.
in Journal of the American Chemical Society
France S
(2017)
Biocatalytic Routes to Enantiomerically Enriched Dibenz[ c , e ]azepines
in Angewandte Chemie
France S
(2018)
Identification of Novel Bacterial Members of the Imine Reductase Enzyme Family that Perform Reductive Amination
in ChemCatChem
France SP
(2017)
Biocatalytic Routes to Enantiomerically Enriched Dibenz[c,e]azepines.
in Angewandte Chemie (International ed. in English)
González-Martínez D
(2020)
Asymmetric Synthesis of Primary and Secondary ß-Fluoro-arylamines using Reductive Aminases from Fungi
in ChemCatChem
Description | Imine Reductases (IREDs) are enzymes that catalyse the reduction of prochiral inexpensive imines (C=N bonds) to optically active or chiral amines (C-N bond) that can be used as intermediates for pharmaceutical synthesis. In this project, we have determined the X-ray crystal structure of imine reductases, leading to conclusions about mechanism and substrate specificity that have informed protein engineering studies. As a result, engineered imine reductases with improved or altered activities have been applied in chemical reactions and also in 'cascade' reactions in which more than one enzyme is employed to turn inexpensive starting materials into chiral amines. We have also demonstrated that a subclass of IREDs catalyses the reductive amination of ketones, a reaction of fundamental importance in organic synthesis that permits the conversion of readily available ketones into chiral amine pharmaceuticals and intermediates. These enzymes, which we have termed RedAms, have also been the subject of structural studies using X-ray crystallography and NMR, and have also been applied in preparative synthetoc reactiions and cascades. |
Exploitation Route | Both imine reductions by IREDs and reductive amination reactions by RedAms have been taken up by industrial groups as a means of synthesising chiral amines. Some of these groups (Roche, AZ, GSK) have published their results and are embarking upon applying these reactions in real industrial processes. In addition, the structural work on IREDs has enabled other academic groups to begin their own protein engineering studies on this class of enzymes. |
Sectors | Agriculture Food and Drink Chemicals Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
URL | https://pure.york.ac.uk/portal/en/researchers/gideon-james-grogan%284ed0bdb4-357d-47f1-8e67-3d81a89a97c9%29. |
Description | CASE Studentship |
Amount | £15,000 (GBP) |
Organisation | AstraZeneca |
Department | Astra Zeneca |
Sector | Private |
Country | United States |
Start | 09/2016 |
End | 09/2019 |
Description | Studentship |
Amount | £90,000 (GBP) |
Organisation | F. Hoffmann-La Roche AG |
Sector | Private |
Country | Global |
Start | 09/2017 |
End | 09/2020 |
Description | Studentship CASE |
Amount | £15,000 (GBP) |
Organisation | Pfizer Ltd |
Sector | Private |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2020 |
Description | Studentship CASE |
Amount | £15,000 (GBP) |
Organisation | Johnson Matthey |
Sector | Private |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2019 |
Description | iCASE BBSRC and Prozomix |
Amount | £90,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2021 |
Title | 8OZW, 8OZV, 8P2J |
Description | Datasets corresponding to structures of the Imine Reductase from Ajellomyces dermatitidis |
Type Of Material | Database/Collection of data |
Year Produced | 2023 |
Provided To Others? | Yes |
Impact | Publication |
URL | https://www.rcsb.org/structure/8OZW |
Title | Imine Reductase from Myxococcus stipitatus V8 variant in complex with NAD+ |
Description | Structure of Imine Reductase from Myxococcus stipitatus V8 variant in complex with NAD+ |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Platform for engineering Imine Reductases |
URL | https://www.rcsb.org/structure/6TOE |
Title | Imine Reductase from Myxococcus stipitatus in complex with NADP+ |
Description | Structure of Imine Reductase from Myxococcus stipitatus in complex with NADP+ |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Platform for engineering Imine Reductases |
URL | https://www.rcsb.org/structure/6TO4 |
Title | Imine Reductase from Streptosporangium roseum in complex with NADP+ and 2,2,2-trifluoroacetophenone hydrate |
Description | Structure of Imine Reductase from Streptosporangium roseum in complex with NADP+ and 2,2,2-trifluoroacetophenone hydrate |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Platform for engineering imine reductases |
URL | https://www.rcsb.org/structure/5OCM |
Title | PDB 5G6R 5A9R |
Description | Coordinates and Structure Factors for Protein Structures 5G6R, 5A9R |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | Structure provides a platform for engineering reductive aminases |
URL | http://www.rcsb.org/structure/5G6R |
Title | PDB 5OJL |
Description | Coordinates and structure factors for imine reductase structures 5OJL |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | No |
Impact | Provides a platform for engineering imine reductases |
URL | http://www.rcsb.org/ |
Title | PDB 6EOD |
Description | Database of structure coordinates and raw electron density from X-ray crystallography experiments |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Provides a platform for the engineering of Reductive Aminase Enzymes |
URL | http://www.rcsb.org/structure/6EOD |
Title | PDB 6EOH 6EOI |
Description | Database of structure coordinates and raw electron density from X-ray crystallography experiments |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Provides a platform for the engineering of Reductive Aminase Enzymes |
URL | http://www.rcsb.org/structure/6EOH |
Description | Manchester Science Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Manchester Science Festival allowed the PDRA and students to host a stand and to present and discuss work with interested members of the general public |
Year(s) Of Engagement Activity | 2016,2017 |
URL | https://www.manchestersciencefestival.com/ |
Description | Press Release |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Press Release on Reductive Aminase Nature Chemistry Publication |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.manchester.ac.uk/discover/news/breakthrough-in-pharmaceuticals-production-with-new-enzyme... |
Description | STEM Teachers' conference |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Two 1 hour workshops on biocatalysis with the aim of raising awareness of current research in biotechnology to those who influence the career choices of children and young adults. A stand was also contributed to an information fair with leaflets about the industrial biotechnology research beacon, the institute and some of its biotechnology and bioenergy networks. |
Year(s) Of Engagement Activity | 2016 |
Description | York Festival of Ideas |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | York Festival of Science allows presentation of a display in a walk-in venue in the centre of York. PDRA on the project was able to present and discuss general aspects of crystallography with general public also with reference to specific project |
Year(s) Of Engagement Activity | 2016,2017 |
URL | http://yorkfestivalofideas.com/2017/ |