(254c) Engineering a Multifunctional Family 5 Glycosyl Hydrolase into a Transglycosidase

Glycans are amongst the most abundant biomacromolecules on the planet but we are far from elucidating their role in the design and regulation of biological systems. There is a dire need of tools to systematically synthesize well-defined glycan polymers as is currently possible with other biopolymers like DNA, RNA, and proteins. Glycans synthesis is naturally performed by membrane associated glycosyl transferases (GTs), but GTs can be challenging to express sometimes for large-scale synthesis. Alternatively, transglycosidases can be selected from a large repertoire of available glycosyl hydrolases (GH) and rationally engineered to enable chemoenzymatic synthesis of designer glycans. Various factors play important roles in engineering a glycosyl hydrolase to function efficiently as a transglycosidases; such as, modulating substrate and product binding site affinities, regulating water accessibility, and enzymatic reaction conditions. Here, we focus on engineering a multifunctional family 5 GH from Clostridium thermocellum (CelE) into a transglycosidase to synthesize diverse glucan-based polymers. Active site mutations and presence of carbohydrate binding modules (CBM) were all found to have a significant impact on the catalytic rate, specificity, and overall mechanism of transglycosylation. All transglycosylation products were characterized using TLC, HPLC, and mass spectrometry to decode the synthesized glycan structures and reaction specificity. We further use molecular dynamics (MD) simulations and small angle X-ray scattering (SAXS) analysis to study the effect of CBM on the mechanism of transglycosylation to rationally design a highly efficient GH 5 based transglycosidase. In summary, we are developing a broadly applicable tool-kit and workflow to rationally convert glycosyl hydrolases into highly efficient and specific transglycosidases.