Award details

Fragment-based activation and modification of industrial enzymes

Reference	BB/N008332/1
Principal Investigator / Supervisor	Professor Roderick Eliot Hubbard
Co-Investigators / Co-Supervisors	Dr John Darby, Professor Gideon Davies, Professor Peter O'Brien
Institution	University of York
Department	Chemistry
Funding type	Research
Value (£)	708,092
Status	Completed
Type	Research Grant
Start date	29/04/2016
End date	28/08/2019
Duration	40 months

Abstract

This project is to provide proof of concept that small molecules can be used to increase the activity of enzymes used for various industrial processes. This builds on our recent discovery and characterisation of an activator for a GlcNAcase enzyme. The hypothesis is that the mechanism of these enzymes includes a change in conformation of the protein; binding of the activators stabilises the transition state, thus increasing the rate of reaction. The small molecules will initially be identified by screening a library of fragments using biophysical methods such as ligand-observed NMR or SPR. Validated hits will be tested in assays for any effect they have on enzyme activity. Selected hits will then be optimised by a combination of structure-guided design (using crystal structures of hits bound to the enzyme) and ligand SAR (from purchase or synthesis and assay of near neighbour compounds). Representatives of enzyme classes used for industrial processes will be studied, with current targets an exoglucanase, endoglucanase, glucosidase and two different amylases. Enzymes have been chosen for which protein production, structure determination and enzyme assay are published and there is evidence that the enzyme mechanism includes a change in conformation. We will attempt to identify activators for at least five enzymes and optimise activators for at least two enzymes. We will also investigate whether covalent attachment of an activator can sustain or improve activation of an enzyme. Initial proof of principle will use our activators of GlcNAcase as a model. Synthesis will use established bioorthogonal chemistry to attach the activator via a cysteine introduced close to the site of activator binding. The modified enzyme can then be used without free activator. Tethering of activator increases the effective concentration which should also increase activity. Finally, we will investigate whether activation of the enzymes changes their product profile.

Summary

Enzymes - large biological molecules that accelerate, or catalyse, chemical reactions - are involved in almost all metabolic processes in living organisms. They have also long been used in many industrial and domestic processes, including as ingredients in washing powders, and in wood and paper processing. More recently their properties are being investigated for use in processes to prepare biofuels. Enzymes work by interacting with other biomolecules to catalyse (speed up) the chemical breakdown of those biomolecules. For some enzymes, this chemical reaction involves changes in the shape of the enzyme, as it switches between an inactivated and an activated state. There is considerable interest in improving the activity of these enzymes, so that they work faster, or so that smaller amounts of enzyme are required for the process (to save cost), or to change the types of molecule produced by the chemical breakdown. The main approach to achieving these improvements is by discovery of new enzymes, either by testing enzymes from diverse sources such as bacteria and fungi, or by making changes (mutations) to existing known enzymes. We have recently demonstrated another approach - finding a small chemical compound (fragment) that additionally binds to an enzyme, stabilising it in its activated state, and thus increasing the speed at which it works. Such compounds could also modify how the enzyme works, changing the types of breakdown products produced. Both of these features (referred to respectively as changes to the enzyme's 'activity' and to its 'product profile') could lead to the development of new commercial products, and of industrial processes that use less energy and convert waste into useful products. There are two main pieces of research in this proposal. The first is to test a number of different enzymes for whether fragments can indeed be identified which change the way the enzyme works. The second is to see if the binding of the fragment can be made permanent to the enzyme. This will mean that the modified enzyme with its attached activating fragment can be used directly in a process, rather than adding large amounts of compound to the reaction mixture.

Impact Summary

The major outcome of this research will be to demonstrate an approach to generating enzymes with increased activity and/or with, possibly, changed product profiles. As well as the academic beneficiaries described elsewhere in this submission, such results would have an impact on the following users of research: 1. Suppliers of industrial enzymes for biofuel production, starch modification, pulp processing and food technology; 2. Scientists and companies generating modified enzymes for carrying out synthesis of fine chemicals and pharmaceuticals. Success in this project would allow researchers and developers in these two areas to develop improved products. Wider society would benefit from improved (and lower cost) products generated by such enzymes which are intrinsically "green" in both their usage of natural materials and the reagents that are used in the processes. In addition, the results could also affect: 3. Pharmaceutical companies who have identified disease conditions where the activity or stability of a particular enzyme or protein in the system needs to be increased. This could provide new approaches to tackling diseases whose effects can be altered by subtly affecting an equilibrium. Examples include cystic fibrosis (by increasing the amount of mutant chloride channel which reaches the membrane surface) and various lysosomal disorders (where specifically increasing the activity of a mutant protein could affect a condition such as Gaucher's disease).

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Industrial Biotechnology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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