Enzymic synthesis of complex carbohydrates using evolved enzymes

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology


Mammalian cells are coated with a vast range of sugar molecules that play important roles in processes ranging from the infection of cells by invading bacteria and viruses, and how the immune system deals with these attacks, to how various cells communicate with each other. Such communication plays vital roles in normal development as well as being affected during disease states such as cancer. There is a growing need to be able to synthesise such sugar molecules for research in a wide range of areas, but complex sugars cannot be cloned, and their chemical synthesis is complicated because of the complex three-dimensional shapes of the molecules themselves. Synthetic chemists have developed routes to some sugar analogues, but these procedures are difficult and not very efficent. Nature's catalysts, the enzymes, carry out such reactions with great precision and efficiency, but their use by man in making complex sugars is limited because the enzymes have evolved to carry out specific reactions, which are not always those that the chemist wishes to carry out. This is particularly true when we wish to make unnatural mimics of the natural sugar. Fortunately, a method of engineering or evolving new, desired enzyme activities in the test-tube is available, and this application seeks funds to use this methodology to alter the properties of two natural enzymes to allow them to carry out the reactions that we want, and to allow them to be used to synthesise the novel complex sugars that we wish. We have already targetted one enzyme, called NAL, to alter its function to make new simple sugars, and have proved the basis of the methodology proposed. Now we will alter two different enzymes (called CNS and ST) to use our new simple sugars to be incoporated into mimics of the complex sugars that are found on the outsides of our cells.

Technical Summary

Carbohydrate-mediated interactions play critical roles in processes as diverse as protein folding, protein trafficking and in mechanisms of infection and immunity in microbe-host interactions. Dissecting the structural features of oligosaccharides which are responsible for these interactions is one of the most challenging areas of cell biology: the carbohydrates involves are (a) complex, (b) generally heterogenous, (c) difficult to prepare using chemical methods, and (d) the products of numerous glycosyltransferases. This grant application will use directed evolution to generate a range of enzymes of general utility in the synthesis of oligosaccharide analogues. Initially, we will develop generic assays for the high-throughput screening of libraries of variants of CMP-N-acetylneuraminic acid synthetases (CMP-NeuAc synthetases; CNSs) and sialyltransferases (STs); critically, the assays will be extremely generic, and may be applied to the evolution of proteins with a wide range of activities. We will then apply the assays in the directed evolution of a range of CNSs and STs which will be of general utility in the parallel synthesis of libraries of oligosaccharide analogues. The substrate specificity of both classes of enzyme will be expanded, and we will optimise and modify the regioselectivity of the STs. The resulting powerful catalysts will then be exploited in the synthesis of a range of complex oligosaccharide mimetics. The work will provide new modified sialic acid containing carbohydrates with uses as diagnostic tools and as potential therapeutics. In the longer term, the evolved enzymes will be of value in the preparation of microarrays of diverse carbohydrates and in the functionalisation of cell surfaces.


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Description We developed a new continuous assay for CTP:Neu5Ac synthetase (CNS) based on detecting and quantifying the PPi released in the reaction and have used it for kinetic studies and in screening directed evolution libraries of CNS variants for new activities and for coupling to novel truncated and substituted sialic acid analogues that were prepared to enable the engineering of sialic acid-processing enzymes.

We have proposed a new mechanism for CNS using site directed mutagenesis of the cloned and expressed enzyme, in which two metal ions play a catalytic role and have identified important residues in catalysis.

We have used directed evolution to improve the activity of CNS for dipropyl containing sialic acid mimetics. We have achieved a 10-fold increase in activity with a 200x switch in specificity.
Exploitation Route Objective 1.
We investigated a series of assays, generating synthetic routes to the required assay compounds. Assays based on pH changes during the reaction are under further development with Fessner (Darmstadt). Detection of the PPi released during the CNS reaction allowed us to produce the first published accurate kinetic data and to elucidate the mechanism involved.

Objective 2. We successfully broadened the specificity of CNS and identified key residues responsible for substrate selectivity and catalysis. A variant (P196S T197W) was identified as having 10-fold higher activity with sialic acid mimetics modified at the glyceryl sidechain.

Objective 3. 9-azido and 9-coumarin sialic acid were synthesized and activated using CNS. Only the CMP-azido compound was a substrate for the cloned sialyltransferase, CstII. Four saturation libraries of CstII were created for screening but dual expression of CNS and CstII in pETDuet vectors was unsuccessful and we did not detect variants of sialyltransferases able to accept these novel substrates.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Our findings impact on the possible construction of complex carbohydrates using enzymes. It has been followed up by our international collaborators.
First Year Of Impact 2013
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Description Innovative Medicines Initiative
Amount € 10,000,000 (EUR)
Funding ID CHEM21 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 11/2012 
End 10/2016
Description iCASE
Amount £94,126 (GBP)
Funding ID BB/L014424/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2017
Description iCASE
Amount £93,520 (GBP)
Funding ID BB/K011448/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
Description COST 
Organisation Technical University of Darmstadt
Country Germany 
Sector Academic/University 
PI Contribution Exchange visit of German scientist to the lab
Collaborator Contribution Exchange visit of German scientist to the lab
Impact Publication by Yi et al
Start Year 2009
Description COST Chemistry Darmstadt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Questions and discussion

Collaborations continued and expanded
Year(s) Of Engagement Activity 2009
Description School visits and talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Questions and answers

Sparked interest in science
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
Description Thackray 
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 a major exhibition for the general public at the Thackray museum (Leeds) celebrating the distinguished career of William Astbury FRS as part of the Royal Society's celebration of its 350th anniversary. As part of the exhibition, ACSMB members will contribute features that highlight scientific links between Astbury's legacy and research in structural biology within the Centre today.

Great outreach and awareness achieved
Year(s) Of Engagement Activity 2010