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Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14 Page 11 of 17

demonstrating the involvement of this residue in fixation of the GlcNAc moiety during catalysis. The
M262G replacement had no remarkable effects on the substrate binding by the enzyme, but considerably
affected the k values, which decreased between 30- and 100-fold. The results suggest that, in addition to its
cat
importance in the hydrophobic patch formation near subsite +1, Met-262 plays an important role in
maintaining the side chain orientation of Glu-160 (the acid/base residue) properly during catalysis, as
suggested by the crystal structure. The replacement of Trp-326 with alanine had various effects on activity.
While it increased the K value by 20-fold and decreased the k value by 13-fold for pNP-Gal, it increased
m
cat
the K values by 1.3- to 4-fold and decreased the k value by 100- to 600-fold for natural substrates. The
m
cat
greater extent of change in the k values for natural substrates than for pNP-Gal indicates the importance of
cat
the sugar-protein stacking interaction at subsite +1 in the transition state. The W326A mutation affected the
activity of LNB and LNT to a similar extent.
Subsite +3: When Arg-327 was replaced with alanine (R327A), a slight increase in the K values for LNT and
m
LNnT was observed without considerably affecting the parameters for the other substrates. Thus, we predict
that the interaction between the reducing-end Glc moiety and Arg-327 may also occur between the protein
and LNnT.

Conservation of the amino acid residues involved in the substrate recognition within structure-
determined GH42 β-galactosidases and within BiBga42A homologs from several Bifidobacterium
species
To date, crystal structures of GH42 members are reported in the apo- and/or Gal-complexed forms only.
The E318S-LNT thus represents the first GH42 structure in complex with a substrate. The subsequent
mutational analysis also identified several amino acid residues important for substrate recognition and
catalysis. We chose all of the above-mentioned amino acid residues and examined their conservation within
the GH42 β-galactosidase members whose crystal structures have been determined [23-30] . Note that the amino
acid residues that interact with the sugar (LNT) through the peptide backbone (Gly-266-Thr-268) were not
considered in the conservation analysis. It should also be mentioned that structural signatures that
discriminate between β-galactosidases and α-arabinopyranosidases within GH42 members have been
[31]
reported previously . Figure 3A shows the conservation pattern of the selected amino acid residues.
Among the 16 amino acid residues, including the two catalytic residues (E160 and E318), 12 residues were
identical among the members. By reflecting distance in the phylogenetic tree [Figure 3B], Bga from
Halorubrum lacusprofundii , β-Gal-ase from Marinomonus sp. , and A4-β-Gal from T. thermophilus
[23]
[30]
[29]
have different amino acid residues at 4 positions (corresponding to residues 24, 156, 221, and 327 of Bi
Bga42A). Those residues are conserved among the three enzymes except for residue 327, which interacts
with the distal reducing-end Glc moiety of LNT in BiBga42A. Unfortunately, as substrate specificities of
these three enzymes have not been reported, we are unable to discuss how these amino acid replacements
are linked with specificity differences among the structure-determined GH42 β-galactosidases. However, we
were aware that there is a striking difference in the substrate specificity between BiBga42A and BlGal42A,
which have identical amino acid residues at the selected 16 positions [Figures 3A and 4A] and share 62%
overall amino acid sequence identity. Both enzymes efficiently hydrolyze β-(1→6)- and β-(1→3)-linkages
and, to a moderate extent, β-(1→4)-linkages when disaccharides were used as substrates [12,25] . However, while
BiBga42A can accept tetrasaccharides, BlGal42A shows very limited activity towards tetrasaccharides. For
example, the catalytic efficiencies (k /K ) of BiBga42A towards 3-galactobiose (Galβ1-3Gal) and 3-
cat
m
galactobiosyllactose (Galβ1-3Galβ1-3Galβ1-4Glc) were 422 and 842 mM s , respectively . In contrast,
[12]
-1 -1
while BlGal42A hydrolases 3-galactobiose with the catalytic efficiency of 52 mM ·s , it was inactive on 3-
-1 -1
[25]
galactobiosyllactose . Moreover, in contrast to BiBga42A, which efficiently hydrolyzed LNT, BlGal42A was
incapable of hydrolyzing LNT . When the active site structures of BiBga42A and BlGal42A were
[12]
superimposed, side chains of the conserved 16 residues were found to have almost the same conformation