Engineering of O-α-Glycoligase to Expand Substrate Repertoire toward Glucose 1-Phosphate

O-α-Glycoligases are mutants of retaining α-glycosidases that lack a catalytic acid/base residue and catalyze the transglycosylation to form α-O-glycosides using α-glycosyl fluorides as donor substrates. This study reports the engineering of an O-α-glycoligase (MalA-D416A) derived from a thermostable α-glucosidase from Saccharolobus solfataricus to use α-glucose 1-phosphate (Glc-1-P) as an alternative donor substrate for flavonoid α-glucosylation. The triple mutant (MalA-D87Q/D416T/A482Y) showed a significant improvement in catalytic efficiency: 25.8-fold for Glc-1-P and 29.5-fold for genistein. Unlike reactions with α-glucosyl fluoride that favor alkaline pH, optimal transglycosylation with Glc-1-P occurred at neutral pH. Due to its hydrolytic activity toward both the donor and the transfer product, and the phosphorylation of the glucosyl moiety in the enzyme intermediate by free phosphate ions, use of a 10:1 molar ratio of donor to flavonoid acceptors in the presence of 30 mM CaCl₂ increased the overall conversion yield from 28.5% for genistein to 54.6-64.6% across six flavonoids tested. Molecular docking analysis indicated that the D87Q mutation improved the binding of Glc-1-P, while A482Y reshapes the acceptor binding pocket to favor effective bond formation of flavonoid 7-hydroxyl groups with the anomeric carbon of the glucosyl moiety in the enzyme intermediate.