Enzymic synthesis of complex carbohydrates using evolved enzymes

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.

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.