|M.Sc Student||Chmelnik Oleg|
|Subject||Protein Engineering of BETA-Xylosidase from GH Family|
52 for Efficient Synthesis of
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
are hydrolytically incompetent glycoside hydrolase (GH) mutants that can
catalyze efficient glycosidic bond formation using activated glycosyl donors
and various acceptors. In these new mutants the catalytic nucleophile is
replaced by a shorter inert residue turning the native GH into inactive
XynB2 E335G (XynB2S) is a nucleophile-deficient mutant of glycoside hydrolase family 52 β-xylosidase from the thermophilic bacterium Geobacillus stearothermophilus. The enzyme exhibits glycosynthase activity by forming glycosidic bond between
α-xylopyranosyl fluoride donor and various aryl sugars. XynB2S can also catalyze the self-condensation reaction of α-xylopyranosyl fluoride, providing mainly α-xylobiosyl fluoride. The glycosynthetic activity of XynB2S was improved by two sequential cycles of directed evolution yielding two variants, V27 and V29, which exhibited an 8- and 35-fold improvement in kcat, respectively. The V29 variant contained 10 amino acid
The objectives of this work were to: (i) identify the amino acid replacements leading to improved glycosynthesis in the XynB2S V29, (iii) determine the kinetic constants of the improved XynB2S variants, and (iii) study and analyze the crystal structure of the XynB2S enzyme and the improved variants.
The three-dimensional structures of XynB2S and the two improved variants, with the xylopyranosyl fluoride donor and the product bound to the active site, were obtained via crystallographic analysis which revealed, together with rigorous kinetic analyses, the crucial amino acids contributing to the improvement of the glycosynthase activity. The XynB2S crystal structure enabled mapping the directed evolution mutations providing insights into the key amino acid replacements and the structural changes that led to enhanced glycosynthesis activity.
Using site directed mutagenesis the mutations situated in vicinity to the active site were introduced to XynB2S glycosynthase to verify their role in glycosynthesis. A new XynB2S F206L T343P double mutant exhibited up to a 100-fold improvement in glycosynthase activity reaching a kcat value of 85±9 sec-1 compared to 0.8±0.1 sec-1 of the parental XynB2S enzyme. Interestingly, the improved variant had higher Kd values toward the donor and the acceptor substrates, suggesting that enzyme-product release could be rate-limiting. To further study this possibility, the effect of viscosity on the kinetic constants of XynB2S variants was determined. If indeed product release is the rate limiting step, kcat should be affected. In the case of XynB2S glycosynthase, the kinetic constants were not affected by changing the solution viscosity ruling out the hypothesis that product release is the rate limiting step for glycosynthase reaction.
Crystal structure analysis of the two mutated residues in the XynB2S F206L T343P variant helped to further understand the elements leading to enhanced glycosynthetic activity. The F206L mutation induce destabilization and migration of a flexible loop that is located near the active site and is suggested to act as a lid over the catalytic pocket. The T343P replacement faces the active site and presumably induces a structural change in the enzyme catalytic pocket caused by the distinctive features of proline, which include conformational rigidity. These changes, affecting the arrangement of a nearby residues, may relate to stabilization of the transition state and enhanced XynB2S glycosynthase activity.