Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Abstract

The Agrobacterium sp. ␤-glucosidase (Abg) is a retaining ␤-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ␣-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (k cat /K m ) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ␣-D-galactopyranosyl fluoride and 4-nitrophenyl ␤-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (؉1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1-3). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4 -6).In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7-9). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the hig...

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

“…However, mutations closer to the catalytic core appear to have a greater effect on the selected catalytic activity (30)(31)(32)(33). These residues for mutagenesis may be selected through a random, rational or combined evolutionary approach.…”

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

“…3 While high yields and selectivities are key features of the glycosynthase reaction, mutagenesis can also play an important role in the improvement and alteration of the catalytic activities of glycosynthases. [4][5][6][7][8][9] Exoglycosynthases, glycosynthases derived from exoglycosidases, have moderate substrate specificity and regioselectivity and can synthesize short chain oligosaccharides (di-, tri-, and tetra-oligosaccharides) that have various glycosidic linkages. [10][11][12] Endoglycosynthases, which are derived from endoglycosidases, generally have high regioselectivity and can catalyze the synthesis of specific glycosidic linkages.…”

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

“…The reaction was first demonstrated with a -glucosidase belonging to glycoside hydrolase family 1 from Agrobacterium tumefaciens. 20) Subsequently, several glycosynthases derived from variousglycosidases have been reported from both exo- [21][22][23][24][25][26] and endo-hydrolases. [27][28][29][30][31][32][33] An -glycoside-forming glycosynthase was also demonstrated with an -glucosidase belonging to GH 31.…”