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.

Publications

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Tavanti M (2017) A biocatalytic cascade for the amination of unfunctionalised cycloalkanes. in Organic & biomolecular chemistry

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Montgomery SL (2017) Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing. in Angewandte Chemie (International ed. in English)

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Mangas-Sanchez J (2017) Imine reductases (IREDs). in Current opinion in chemical biology

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Lenz M (2018) New imine-reducing enzymes from ß-hydroxyacid dehydrogenases by single amino acid substitutions. in Protein engineering, design & selection : PEDS

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Grogan G (2018) Synthesis of chiral amines using redox biocatalysis. in Current opinion in chemical biology

 
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 The findings have been used by pharmaceutical companies as a basis upon which to embark on research programmes designed to use the enzymes in processes for the synthesis of pharmaceutical intermediates. Some of this interest is evidenced in publications by these companies.
First Year Of Impact 2016
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description CASE Studentship
Amount £15,000 (GBP)
Organisation AstraZeneca 
Department Astra Zeneca
Sector Private
Country United States
Start 10/2016 
End 09/2019
 
Description Studentship
Amount £90,000 (GBP)
Organisation F. Hoffmann-La Roche AG 
Sector Private
Country Global
Start 10/2017 
End 09/2020
 
Description Studentship CASE
Amount £15,000 (GBP)
Organisation Johnson Matthey 
Sector Private
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Studentship CASE
Amount £15,000 (GBP)
Organisation Pfizer Ltd 
Sector Private
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description iCASE BBSRC and Prozomix
Amount £90,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
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/