# Unveiling the Transgalactosylation Switch of a GH42 β‑Galactosidase from the Infant Isolate Bifidobacterium breve DSM20213

## TL;DR

Scientists discovered a key amino acid in a bacterial enzyme that controls whether it produces prebiotic sugars or breaks them down, which could improve infant formula production.

## Contribution

The study identifies Arg121 as a critical switch controlling transgalactosylation versus hydrolysis in GH42 β-galactosidases and demonstrates how modifying it enhances prebiotic sugar production.

## Key findings

- Arg121 in Bbreβgal-III influences the enzyme's preference for hydrolysis over transgalactosylation.
- Mutating Arg121 to cysteine (R121C) increased GOS yield from 17% to 34%.
- Modifying the water tunnel in the enzyme's active site also boosted transgalactosylation activity.

## Abstract

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.