Unveiling the Transgalactosylation Switch of a GH42 β‑Galactosidase from the Infant Isolate Bifidobacterium breve...

β-Galactosidases catalyze the transgalactosylation of lactose to produce prebiotic galacto-oligosaccharides (GOS), key fortificants in infant formulas. A β-galactosidase from the infant isolate Bifidobacterium breve DSM20213 (Bbreβgal-III), which belongs to the glycoside hydrolase (GH) family 42, exhibits limited transgalactosylation activity, resulting in a low yield of GOS with the predominant formation of β-(1→6)-linked GOS. Structural analysis revealed a hydrophobic-rich active site and the presence of a water tunnel connecting the deeply buried active site to the exterior environment. The highly conserved hydrophilic Arg121 residue, which is adjacent to the catalytic acid/base residue Glu160, was found to play a crucial role in the hydrolytic activity of Bbreβgal-III. The guanidino group of the Arg121 side chain forms a network of hydrogen bonds involving the catalytic Glu160 and a water molecule in the water tunnel. This influences the coordination environment of the active site, resulting in a preference for hydrolysis over transgalactosylation in Bbreβgal-III. Site-saturation mutagenesis at Arg121 revealed that all variants enhanced the transgalactosylation activity. Among these, Bbreβgal-III-R121C exhibited the highest GOS yield, reaching 34% mass of total sugars in the transgalactosylation reaction compared to 17% by the wild-type enzyme. Bbreβgal-III-R121C not only enhanced transgalactosylation but also displayed distinct changes in the main GOS components synthesized, including a shift from 6'-galactosyllactose, which is formed predominantly by wild-type Bbreβgal-III, to 3'-galactosyllactose and a notable increase in the formation of β-(1→2)-linked GOS. These results suggest that Arg121 acts as a key switch for transgalactosylation/hydrolysis activity in GH42 β-galactosidases. Furthermore, water tunnel engineering, i.e., modification of the active-site access pathway, using alanine scanning also increased the transgalactosylation activity. Disrupting the movement of water molecules within the tunnel resulted in higher transgalactosylation activity. Understanding the catalytic importance of amino acids involved in transgalactosylation and rational mutagenesis of active-site residues provided further insights into the structure-function relationships of β-galactosidases within the GH42 family.