A mutant glycosidase for oligosaccharide synthesis S.M. Hancock, K.E. McAuley 1 and B.G. Davis Chemistry Research Laboratory, University of Oxford, Mansfield Road, OX1 3TA Oxford, UK 1 Diamond Light Source, Chilton, OX11 0DE Didcot, UK Oligosaccharides play key roles in biological recognition and signalling mechanisms and in recent years there has been a growing interest in the development of carbohydrate-based drugs. 1-5 The potential therapeutic role of carbohydrate-based drugs has so far been underexploited, largely due to the complexities in their chemical synthesis. Carbohydrate-processing enzymes can be used as an alternative to chemical synthesis and have the advantage that the reactions they catalyse are regio- and stereo- specific. 6 We are exploring the creation of unique glycosidase catalysts, compatible with high temperature and organic solvents, which will form glycosidic linkages between any two monosaccharides in one step. We have been investigating glycoside formation with the retaining -glycosidase from Sulfolobus solfataricus (Ss G). 7, 8 Ss G is thermophilic, and displays tolerance to organic solvents. These attributes highlight the potential of this enzyme as a universal glycosylation catalyst. With the prospect of creating more versatile catalysts for the carbohydrate chemist's toolkit, we explored the redesign of the enzyme's catalytic machinery by mutagenesis, targeting enzyme mechanism. Figure 1. Ss G, a Family 1 glycosyl hydrolase (pdb: 1gow). 9 The active site nucleophile and general acid/base residues are shown in blue and red, respectively. Family 1 glycosyl hydrolases contain two glutamates in the active site, one of which acts as the catalytic nucleophile and the other as general acid/base. Using rational mutagenesis, a mutant of Ss G has been constructed that synthesises oligosaccharides in excellent yields (UK priority patent application no. 0329011.1). We have been using a combined approach of X-ray crystallography, MS trapping experiments and kinetic studies in order to understand the mechanism by which the enzyme catalyses this process. 61