EpiSwitch: the specificity of carbohydrate epimerases

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.

Carbohydrates are essential for life. Structurally, they are the most diverse class of chemical compounds in nature. Monosaccharides are the basic building blocks of larger carbohydrate structures, such as oligosaccharides and glycans. Common monosaccharides, such as D-glucose, obtain from de novo carbohydrate biosynthesis of the cell or are incorporated from nutrients. Less common monosaccharides, such as D-galactose and many others, are generated from a core set of precursor monosaccharides. Epimerization (chemically: the configuration change at a single stereocenter of diastereomers) is a biologically important and synthetically promising transformation for the direct interconversion of monosaccharide structures. In biology, epimerization happens typically at the level of the nucleotide-activated sugars. The enzymes promoting epimerization (epimerases) involve unique mechanistic principles of chemical and biological catalysis. Determinants of reaction selectivity and substrate specificity are not well understood for epimerases. The discovery of new, natural or engineered, epimerases with an expanded scope of sugar nucleotide substrates used is important to promote the mechanistic inquiry and can facilitate the development of new enzyme applications. The EpiSwitch project has investigated sugar nucleotide epimerases and enzymes (decarboxylases) related to them by structure, mechanism and function. Joint research of the partners from Belgium and Austria has resulted in the discovery and characterization of a new thermostable C2 epimerases that catalyzes the D-glucose/D-mannose interconversion in all nucleotide-activated forms of D-glucose. The enzyme has enabled new approaches to the study of the epimerization mechanism and can be a useful tool for biocatalytic synthesis. Study of UDP-glucuronic acid C4 epimerase and related UDP-glucuronic acid decarboxylases has resulted in important discoveries that contribute to a major advancement of the field. (1) The mechanism of C-branched UDP-apiose biosynthesis was uncovered through elucidation of structure and catalytic function of the UDP-xylose/UDP-apiose synthase from the plant Arabidopsis thaliana. (2) The mechanism of C4 epimerization of UDP-glucuronic acid was revealed through detailed biochemical characterization and structure study of a bacterial epimerase from Bacillus cereus. (3) Using experimental and computational approaches, the catalytic pathway of the UDP-glucuronic acid C4 epimerase was elucidated. Engineered epimerases are converted to primitive UDP-glucuronic acid decarboxylases that synthesize UDP-xylose, demonstrating a first case of selectivity switch in this class of enzymes. (4) A new mechanistic hypothesis is proposed: epimerases and decarboxylases employ stereo-electronic control to achieve product selectivity in their catalytic reactions.