|Ph.D Student||Ben-David Alon|
|Subject||Engineering Glycoside Hydrolases for Novel|
Applications and Properties
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
The objectives of this research were to analyze the crystal structure of the glycoside hydrolase family 43 b-xylosidase XynB3, to characterize the glycosynthase activity of the family 52 b-xylosidase XynB2, and to improve to glycosynthase activity of XynB2 via directed evolution.
XynB3 is an inverting family 43 β-xylosidase from the thermophilic bacterium Geobacillus stearothermophilus T-6. Each XynB3 monomeric subunit is organized in two domains: an N-terminal five-bladed β-propeller catalytic domain, and a β-sandwich domain. The active-site of the enzyme possesses a pocket-topology, which correlates well with its exo mode of action. Structures of the enzyme-substrate complex provided insights into the role of the three catalytic residues found in all family 43 glycosidases. The general base, Asp15, is located 5.2 Å from the anomeric carbon, which enables it to activate a water molecule for a single-displacement attack on the anomeric carbon. Glu187 is 2.4 Å from the glycosidic oxygen and can protonate the leaving aglycon. The third catalytic carboxylic acid, Asp128, is 4 Å from the general acid; modulating its pKa and keeping it in the correct orientation relative to the substrate. In addition, Asp128 plays an important role in substrate binding via the 2-O of the glycon, which is important for the transition-state stabilization. Taken together, these key roles explain why Asp128 is invariant among all five-bladed β-propeller glycosidases.
Glycosynthases are mutant glycosidases engineered to catalyze glycosylation reactions using glycosyl fluoride as a sugar donors. In this study we demonstrated that XynB2E335G from G. stearothermophilus, can function as an efficient glycosynthase, transferring a-D-xylopyranosyl fluoride donor to various acceptors. The enzyme can also catalyze the self-condensation reaction of a-D-xylopyranosyl fluoride, providing mainly a-D-xylobiosyl fluoride. When the XynB2E335G was combined with a glycoside hydrolase family 10 glycosynthase, high molecular weight xylooligomers were obtained using a-D-xylopyranosyl fluoride as the sole substrate.
To improve the activity of XynB2E335G via directed evolution we developed a general high-throughput screening procedure for glycosynthase activity, which is based on the release of hydrofluoric acid, a by-product of all glycosynthase reactions. This assay is sensitive, does not require the synthesis of special chromophoric or modified substrates, and most importantly, is applicable for all glycosynthases. Using this novel screening procedure on error-prone PCR libraries, we isolated improved glycosynthase variants exhibiting up to 35-fold improvement in kcat value. Interestingly, the improved variants had higher KM values towards the acceptor and the donor, suggesting that enzyme-product release is rate-determining for kcat.