FWF - EpiSwitch - Characterization and engineering of the specificity determinants of carbohydrate epimerases to achieve a specificity switch

Project: Research project


Carbohydrates are the structurally most diverse class of chemical compounds in nature. The function of carbohydrates in biology is likewise diverse but one of their key roles is in biological recognition where the interactions of other biomolecules (e.g. proteins) with carbohydrates are essential for specificity. Carbohydrate-based recognition pervades all forms of life and it is manifested in subcellular processes as well as in the complexity of humans physiology under a state of health or disease. The primary level of structural diversity in carbohydrates is constituted by the various monosaccharides formed in cellular biosynthesis. Starting from simple precursors such as UDP-D-glucose, the naturally activated form of "dextrose", structural variation is achieved by means of enzymatic epimerisation. The term epimerisation means that the configuration at a certain stereogenic center, say carbon 4 of D-glucose, is inverted to give a new carbohydrate structure, in this case D-galactose. Many rare sugars are made in biology using epimerisation chemistry and they are also of interest for application in health-related foods, in cosmetics and also in medicine. Although on the surface of it epimerisation seems to be a very simple chemical transformation, it is complicated to achieve indeed. A chemical synthesis would require multiple steps involving an extensive amount of activation and protection/deprotection chemistry. The enzymes catalyzing epimerisations in biology are called carbohydrate epimerases (CEP) and they do so with remarkable specificity. CEPs have recently been classified according to their structure and mechanism into groups, that were called CEP families. It was found that the major enzyme family, termed CEP 1 family, comprised a remarkably broad diversity of reactivities and specificities. Because differences in the functional properties of members of family CEP1 have evolved on the basis of an overall conserved protein structure and catalytic mechanism, a research question of particularly high significance appeared to be the elucidation of active-site structural variation in CEP enzymes in relation to their catalytic function and specificity. The groups of Tom Desmet (Ghent University) and Bernd Nidetzky (Graz University of Technology) joined to address this problem in the current project. Using complementary expertises in the two groups, they propose to use advanced mutational analysis combined with detailed kinetic and mechanistic characterization of variant CEPs to characterize and thus unravel the molecular determinants of CEP specificity and reactivity. Besides establishing the important structure-function relationships for CEP enzymes they would like to use the knowledge gained in the research to demonstrate that a specificity switch can be achieved in an existing CEP enzyme. A successful project would provide fundamental insights into an important group of carbohydrate-active enzymes and could be useful to provide new enzymes for rare sugar synthesis.
Effective start/end date1/03/1728/02/20