Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

Abstract

23Trends in Glycoscience and Glycotechnology Vol. 21 No. 117 (2009) AbstractReducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (X1) from its reducing end. Rex hydrolyzes a-xylobiosyl ‰uo-ride (a-X2F) to yield xylobiose (X2) only in the presence of X1, conˆrming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X 3 ) from a-X 2 F and X 1 . However, F -releasing activities of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F -releasing activity from a-X2F in the presence of X1. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting a-1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact.

Enzymes in the Synthesis of Glycoconjugates

“…Three classes of enzymes are employed for carbohydrate synthesis: GTases, exo -glycosidases, and endo -glycosidases. Additionally, these classes of enzymes have been enhanced over the past decade through genetic engineering to form mutant enzyme classes, such as glycosynthases and thioglycoligases, which are designed or selected for increased yields, enzyme stability, and improved donor and acceptor specificity. ,, Enzymes can also be exploited in the glycoengineering of cells (see section 5).…”

“…Detailed accounts of evolving and applying glycosynthases have been provided by Withers and co-workers. ,,,, Notably, this mechanism-based engineering technique has been expanded to create novel thioglycosynthases . Glycosynthases have also been more recently generated from the inverting glycosidases, including exo -β-oligoxylanase , and 1,2-α-fucosidase, , expanding the types of glycosidases that can be engineered for synthetic ends. Undoubtedly, engineering new glycosynthases will continue to create useful enzymes for glycan synthesis, and the current success of the technique is underscored by the variety of enzymes that are available to enact various stereo- and regiochemical linkages, as summarized in Table . ,− …”

“…The need to understand glycans and glycoconjugates has motivated an extraordinary amount of work toward developing synthetic and chemoenzymatic methods to produce them in a pure form over the past ten years. While many of these routes have been the subject of focused reviews, − this account provides a thorough resource of the chemoenzymatic and whole-cell tools that are available for producing glycans and glycoconjugates with defined structures. Because the covered topics are broad, succinct discussions of key concepts are provided in the text along with a few representative examples, while more in-depth consideration of subjects can be gained by consulting the extensive sources to the primary literature provided throughout.…”

Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity

“…Recent reports have shown that inverting glycosidases may be converted into glycosynthases . Glycosynthases are engineered glycoside hydrolases that catalyze the synthesis of glycosides from activated donors, such as glycosyl fluorides or glycosyl azides . A GH8 enzyme, reducing‐end xylose‐releasing exo‐oligoxylanase from Bacillus halodurans ( Bh Rex), is an inverting glycosidase that has the (α/α) 6 barrel fold and may be converted to a glycosynthase .…”

Structural Similarity between a Starch-hydrolyzing Enzyme and an N-Glycan-Hydrolyzing Enzyme: Exohydrolases Cleaving α-1,X-Glucosidic Linkages to Produce β-Glucose

“…Recently, the group of Wada et al has reported that some mutants of GH95 1,2-α-L-fucosidase showed 1,2-α-Lfucosynthase activity (46), suggesting that enzymes of families GH15, GH37, GH63, and related enzymes can be converted into glycosynthases by means of mutagenesis (51,52). Further studies may provide information on the industrial application of these enzymes.…”