Enzymic synthesis of complex carbohydrates using evolved enzymes
Lead Research Organisation:
University of Leeds
Department Name: Inst of Molecular & Cellular Biology
Abstract
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.
People |
ORCID iD |
Alan Berry (Principal Investigator) | |
Adam Nelson (Co-Investigator) |
Publications
Bolt A
(2008)
Directed evolution of aldolases for exploitation in synthetic organic chemistry.
in Archives of biochemistry and biophysics
Yi D
(2013)
Engineering of a Cytidine 5'-Monophosphate-Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids
in Advanced Synthesis & Catalysis
Horsfall LE
(2010)
Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.
in The FEBS journal
Campeotto I
(2009)
Structure of an Escherichia coli N-acetyl-D-neuraminic acid lyase mutant, E192N, in complex with pyruvate at 1.45 angstrom resolution.
in Acta crystallographica. Section F, Structural biology and crystallization communications
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 | 09/2013 |
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 | 09/2014 |
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 | 2007,2008,2009,2010,2011,2012,2013,2014 |
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 |