Abbreviations: α-GalF, α-galactopyranosyl fluoride; Gal-Lac, β-lactosyl α-D-galactopyranoside; 20 GH, glycoside hydrolase family; HMBC, heteronuclear multiple bond correlation; 21 high-performance anion exchange chromatography-pulsed amperometric detection. The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-5 established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases 6 derived from α-glycosidases can give poor yields of desired products because they require 7 generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed 8 by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-9 galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant 10 enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer 11 from α-galactosyl fluoride with assistance of external anions. Formate was more effective at 12 restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the 13 presence of the azide is because of low activity of the ternary complex between enzyme, β-14 galactosyl azide, and acceptor. A three-dimensional structure of the mutant enzyme in complex 15 with the transglycosylation product, β-lactosyl α-D-galactoside, was solved to elucidate the 16 ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2, and 3 of 17 the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside 18 hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α 19 loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial 20 hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside 21 hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside-22 recognition is conserved among GH27, 31, 36, and 97 α-galactosidases. 23 24

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