Structure and function of archetypal glycosyltransferases

Simple and complex carbohydrates have been described as `the last frontier of molecular and cell biology`. The carbohydrates, or often `the sugars`, are biomolecules characterised by enormous structural complexity and functional diversity. To a wider public, they are known mainly because they provide a major caloric portion of the human diet and sometimes impart a sweet taste to the product. However, physiological roles in which carbohydrates act as a `cellular language` have been unveiled and rely on the huge coding potential of the individual monosaccharides that constitute the functional structure. Among the plethora of carbohydrate-active enzymes, those which can catalyse the formation of specific linkages between the monosaccharide building blocks to yield oligosaccharides are especially challenging. This group of enzymes, functionally classified as `the glycosyltransferases` (GTs), is large, and its members differ in respect to both amino acid sequence and three- dimensional structure, partly reflecting the complexity of the reaction products of their catalytic action. Obviously, a very well-defined orchestration of the action of different GTs in a place and time-dependent context is required to achieve a tight regulation of carbohydrate-mediated cellular responses at all levels of metabolism. In order to harness fully the newly bequeathed genomic resource in the form of a myriad of open-reading frames whose translation products that are likely involved in oligosaccharide synthesis, modification and turnover, we need to understand better how GT sequences relate to enzyme structure, mechanism, and specificity. The project aims at unraveling some "sweet secrets" of GT structure-function relationships by focusing on a representative enzyme group within the GT class: the phosphorylases. We will use the genome of the archaeon Sulfolobus solfataricus as resource of novel and seemingly archetypal variants of glycogen phosphorylase and trehalose phosphorylase, two important GTs of energy-related carbohydrate metabolism. The archaeal enzymes and selected mutants thereof will be examined through detailed biochemical and mechanistic characterisation, and their properties compared with homologues seen in other organisms and cell types. Through this process, means for extrapolation of molecular information to other sequence-related GTs are provided and strategies for the utilisation of the glycogenomic resource inspired.

The carbohydrates, or often `the sugars`, are biomolecules characterized by enormous structural complexity and functional diversity. To a wider public, they are known mainly because they provide a major caloric portion of the human diet and sometimes impart a sweet taste to the product. However, physiological roles in which carbohydrates act as important elements of biological recognition have been unveiled and it was shown that they rely on the huge coding potential of the individual monosaccharides that constitute the functional structure. Among the plethora of carbohydrate-active enzymes, those that can catalyse the formation of specific linkages between the monosaccharide building blocks to yield oligosaccharides are of special interest. This group of enzymes, functionally classified as `the glycosyltransferases`, is large, and its members differ in respect to both amino acid sequence and three-dimensional structure, partly reflecting the complexity of the reaction products of their catalytic action. Phosphorylases constitute a special group of glycosyltransferases that are widely distributed in microorganisms. Recent studies have indicated that phosphorylases are very useful catalysts for oligosaccharide synthesis in glycobiotechnology. In this project, different phosphorylases were characterized with respect to their biochemical properties and the catalytic mechanism utilised. The results of the project made an important contribution to the development of a biocatalytic process for production of glucosylglycerol, a naturally occurring molecule that is now applied as active ingredient in cosmetics.