Exploring, evolving and exploiting coupled racemase/acylase biotransformation systems.

Modern medicine has used drugs to cure disease, alleviate chronic pain and increase life spans. Drug companies must make drugs under very strict regulations - these have to be 100% pure. One complication is that Nature has evolved to make some chemicals look the same, weigh the same, are made of the exact same atoms but they are different in the fact that they are mirror images of each other - like a pair of hands - these are called "enantiomers". It turns out that Nature tends to work with only one enantiomer and it is often observed that the opposite one is toxic, this is the case with drugs, we desire only one enantiomer. Important types of natural molecules where handed-ness make a big impact are amino acids. These are the building blocks of the proteins inside every cell. Proteins are made up hundreds and thousands of amino acids, polymerised together head-to-tail like beads on a string. These chains fold up into specific 3 dimensional shapes that can carry out many essential functions in the cell. Enzymes are also proteins and they are the workhorses of the cell - enzymes are tiny catalysts that speed up the conversion of molecule A to molecule B. Without an enzyme these conversions would take years but an enzyme catalyst can accelerate the speed of a reaction over 10 billion times. Enzymes allow us to breakdown our food, provide us with energy and help us repair damaged tissue. It turns out that these enzymes can also be put to work to make the very molecules that drug companies want. Enzymes are very specific and only work with one particular hand/mirror image of a molecule. Pharmaceutical companies endeavour to make large amounts of drugs the cheapest, purest and least wasteful way they can. Drugs are complicated molecules, many are made from amino acid building blocks (only one mirror image) in a multi-step process. Because the process uses only one of the mirror images of the starting material, the other mirror image is not used and in thus 50% is wasted.
Our project aims to tackle this fundamental problem. We aim to make key amino acid building blocks of only one hand or another and use up 100% of the starting material. We will use enzymes to carry out the conversion of amino acid precursors to the target amino acid. The enzymes themselves were not designed for this specific job so we have to engineer the enzymes at a molecular level. We can do this by rational design - with knowledge of the molecular structure we can make specific changes and hope that the new enzyme will have the desired characteristics - speed, efficiency and stability. We can also carry out a random approach then fish out the desired new enzyme from the mixture. The enzyme we study catalyses the interconversion of the mirror image of one amino acid precursor into the other mirror image - this is called a racemase. Once we have the ideal racemase we will pair it up with another enzyme - an acylase - this one converts the amino acid precursor into the final amino acid but is specific for only one of the mirror images. So, we will start with both starting amino acid precursors - 50% of each mirror image. The acylase will convert one half into the product until it is used up; at the same time the racemase will be doing its job converting the unused precursor into its mirror image and when this happens the acylase can convert it. In a perfect world all of the precursor will be used up (100% conversion) and there will be no precursors left. Moreover, the enzymes can be produced cheaply, re-cycled, are bio-degradable and they work in water. We have already made good progress and now require funds to optimise the whole process. We will do this at University in partnership with a company that are experts in making amino acid precursors and products for the pharmaceutical industry. As well as making valuable tools for drug production we will also gain fundamental knowledge about enzyme design that others can apply to numerous useful processes.

The use of enzymes in biocatalytic transformations is an attractive way to produce chemical intermediates for the pharmaceutical industry by low cost, green methods, but the bottleneck is that natural enzymes with suitable substrate specificities and catalytic rates are not available. This industrial/academic partnership aims to develop a biocatalysis "toolbox" for the production of enantio-pure amino acids using novel, engineered bacterial N-acetyl-amino acid racemases (NAAARs, members of the enolase superfamily) in combination with L- or D-acylases. This combination permits a Dynamic Kinetic Resolution (DKR) of racemic starting materials. Wild-type NAAAR from Amycolatopsis has low catalytic rates with N-acetyl L-/D-methionine but we used mutagenesis and screening to isolate a double mutant enzyme (NAAAR G291D F323Y) with improved catalytic rates. We will further explore the NAAARs by making more mutants and use other bacterial NAAARs to make chimeras. We specifically want to generate novel NAAARs with activity towards substrates with bulky side chains such as N-acetyl-tert-Leucine. These, and non-natural amino acids, are in demand by the pharma industry since they are found in many drugs. We aim to develop an amino acid oxidase (AAO) coupled assay to move towards a high throughput NAAAR assay and use a chiral HPLC instead of the agar plate assay. The x-ray structures of novel NAAARs will help us understand and predict the molecular basis of increased catalytic rates. With a suite of NAAARs we wish to determine if there is any correlation between NAAAR and enolase activities. We aim to optimise the compatibility of the NAAAR/acylase DKR by combining these in scale-up biotransformations. We will generate tools of use to our industrial partner and others. The knowledge gained will also be of interest to academics working in the biocatalysis, enzyme mechanism and protein engineering fields.

The proposed project maps directly onto BBSRC priority areas in the area of 'Technology development for bioscience'. Underpinning the programme of work is the need to create general toolkits for industrial biocatalysis using robust and adaptable protein scaffolds/frameworks. We have progressed beyond 'proof of principle' stage; the PDRA will now develop our new technology to evolve a suite of new biocatalysts that are important in industrial biotransformations and which are not yet available in the commercial or academic sectors. Results will inform a broad range of academics with interests in the fields of biochemistry, chemical synthesis, biocatalysis, enzyme mechanism, protein structure, biotechnology, synthetic biology and the origins and evolution of enzyme catalysis. Furthermore, the results and methods will provide support and validation for the use of enzymes in "greener" processes.
Benefits:
Academic partner: This collaboration will enable the academic partners to work towards creating commercially important chiral building blocks. It is crucial that the novel biocatalyst development is carried out with industrially relevant target molecule(s) in mind. Through a field of use licence it maybe therefore possible for the UoE to benefit from the collaboration for an agreed return, the academics will also benefit from the interaction with the company supervisors to aid understanding of commercial realities behind developing new enzyme-based processes. It is important for academics to operate in the industrial environment. Also, the School of Chemistry has a highly successful MChem Masters programme with a year in Industry. We have ~60 students out on placement every year at ~30 partners and many of our graduates gain employment with our industrial partners. It is crucial that academics maintain current links and expand their contacts - we will bring Dr. Reddy's onboard so undergraduates will benefit from this exciting science. In a reciprocal arrangement, we also use industrial partners to deliver lectures to undergraduates (e.g. Medicinal Chemistry) and Dr. Reddy's can join the team to deliver cutting edge successful (and failed!) case studies. As well as generating novel IP, it is important that the PI and industrial partner publish their work in high impact, high profile publications (JACS, Angew. Chemie, Green Chemistry) for the benefit of the academic community. We will also contribute to international meetings, both academic and industrial-focus groups.
Industrial partner: Dr. Reddy's Laboratories is a global pharmaceutical company whose purpose is to provide affordable and innovative medicines. Dr. Reddy's wholly owned subsidiary, Chirotech Technology Ltd, Cambridge, serves as a centre of excellence and R&D facility for asymmetric organic synthesis, developing novel processes and providing chiral synthons for active pharmaceutical intermediates (APIs). Chirotech Technology has pioneered of the use of enzymes and microorganisms in the manufacture of chiral pharmaceutical intermediates. Chirotech will benefit from using thus generated portfolio of commercially valuable enzymes, possessing desired properties like extended substrate selectivity profile, both in terms of substrate structure, high-activity and stability at industrially relevant substrate concentrations. The development of novel biocatalysts would help Chirotech to design innovative and affordable bioprocesses for API manufacture. The enzymes will be made available under CDAs and MTAs to the collaborating company enabling exploitation of the methodology. The company also benefit from using facilities and working closely with partners at a ScotChem institute (EastChem (UofE, St. Andrews) was rated 4th overall at the last RAE). Dr. Reddy's have expanded their presence at the Cambridge site and wish to make strong collaborations with UK academics. This is good news in the current climate of recent UK site closures from important players in big pharma.