Reversing Glycoside Hydrolases into Glycosynthases for Efficient Enzymatic Synthesis of Oligosaccharides

Conversion of glycosyl hydrolases (GHs) into glycosynthases provides an attractive approach for efficient enzymatic synthesis of complex carbohydrates. Such rational was recently applied to GsXynB2, a retaining GH52 β-xylosidase from the thermophilic bacterium Geobacillus stearothermophilus, which efficiently hydrolyzes di-xylose sugars into their xylose monomer components. We were able to reverse the catalytic reaction of GsXynB2 from hydrolase to glycosynthase, synthesizing di-xylose-F sugars from xylose-F reactants, by a single mutation, changing the catalytic nucleophile from E335 into G335. This reverse activity was then improved 35-fold upon introduction of 10 additional random mutations, obtained through two cycles of directed-evolution experiments. Activity characterization of different GsXynB2 glycosynthase variants, possessing different combinations of these random mutations, revealed that the glycosynthase activity can be improved by two orders of magnitude, by only two of these mutations, T343P and F206L. In order to understand the structural basis for this improvement, we determined the 3D structures of a series of different GsXynB2 variants by X-ray crystallography, alone and in complex with their sugar substrates and products, allowing observation of different snapshots along the reaction coordinate. In this talk, we will show the results obtained from these studies, which together with complementary molecular dynamics (MD) simulations, suggest that the improved glycosyntase activity is due to an increased flexibility around the active site. As will be demonstrated, these results may also be generalized for the rational design of improved glycosynthases of many other specificities and activities, for the efficient and relatively inexpensive synthesis of a wide range of carbohydrates.