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Page 10 of 17 Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14

side chain of Glu-366 changes its conformation upon the substrate binding and makes a hydrogen bond
with the O6 atom of the Gal moiety. It is unclear whether the difference between the Gal molecules in the
two crystallographic structures is caused by an artifact during crystallization or reflects dynamics that could
occur during catalysis. Nonetheless, it is noteworthy that the Gal moiety in the E318S-LNT structure adopts
a slightly distorted H -like (ψ ~ 324°) conformation. In the case of GH2 LacZ β-galactosidase from
O
5
Escherichia coli, the Gal moiety of allolactose bound to subsite -1 took the H (ψ ~ 210°) conformation .
3
[49]
4
The β-galactosidic bond of allolactose in LacZ was in a pseudo-axial orientation that is suitable for the
3
4
nucleophilic attack from the catalytic Glu residue, and a conformation itinerary through C - H - C was
4
4
1
1
proposed for the catalysis. Since it has been suggested that sugar distortion is generally involved in the
catalysis of β-glycosidases , our observation of the sugar distortion in BiBga42A might suggest that a
[50]
similar conformational change occurs during catalysis in GH42.
The leaving group trisaccharide moiety [lacto-N-triose II (LNT2), GlcNAcβ1-3Galβ1-4Glc] of LNT is
recognized by the protein via both stacking and hydrogen bonding interactions. The sugar ring of the
GlcNAc in subsite +1 forms a stacking interaction with the side chain of Trp-326 [Figure 2B]. The nitrogen
atom of the N-acetyl group is recognized by a direct hydrogen bond with the carboxyl group of Glu-160 (the
catalytic acid/base). The N-acetyl group is fixed by a hydrophobic patch created by the side chain of Met-
262 and by an intramolecular hydrogen bond with the O2 atom of the Gal moiety of the Lac unit. The C6
hydroxymethyl group of the GlcNAc moiety, which faces solvent, is constrained in a gg conformation by a
packing interaction with Phe-221 from the neighboring molecule of the trimer. Phe-221 is located on the tip
of a long helix next to Trp-200 [Figure 1]. As for the Gal moiety of the Lac unit, no specific interaction with
the protein was observed [Figure 2C]. In addition, the reducing-end Glc is recognized by two direct and one
water-mediated hydrogen bonds. The direct contacts are formed between the O6 atom of the Glc moiety
and the side chain of Arg-327 and between the O2 atom of the Glc moiety and the carbonyl oxygen of the
peptide bond of Gly-266-Cys-267. Water-mediated hydrogen bonds from the O3 atom of the Glc moiety are
formed with the nitrogen atom of the peptide bond of Cys-267-Thr-268. The Gal and Glc moieties of the
Lac unit of LNT show higher B-factors than the Gal and GlcNAc moieties of the LNB unit of LNT,
suggesting that the reducing-end Lac unit is relatively flexible [Supplementary Table 1]. Overall, the LNT
molecule bound to the catalytic pocket of the E318S mutant is recognized by an extensive hydrogen bond
network and several hydrophobic interactions which involve a residue from the neighboring subunit.

Mutational analysis
We then introduced amino acid replacements into several positions described above to evaluate their
importance in the reaction process. The substrates used were pNP-Gal, LNB, LNT, and LNnT. The values
[11]
obtained for the WT enzyme were comparable with those reported in our previous study .

Subsite -1: Alanine substitution for Arg-121 (R121A) increased the K values for the four substrates by 2- to
m
3-fold, while it reduced the k values by 20- to 30-fold [Table 1]. The results suggest that Arg-121
cat
contributes more to catalysis than to substrate binding. The Y287F substitution, which presumably causes
the loss of a hydrogen bond with the Gal moiety of LNT, did not cause a marked alteration in the K values
m
but caused a marked reduction in the k values by 80- to 170-fold. The effect on the k values for pNP-Gal
cat
cat
was the third largest, following the nucleophile mutation (E318A) and the acid/base catalyst mutation
(E160A), indicating that this residue plays a crucial role in the catalysis.
Subsite +1: Alanine substitution for Phe-221 (F221A), the residue from the neighboring subunit, slightly
increased the K values for all substrates within a range less than 3-fold and decreased the k values by 2- to
cat
m
20-fold. The considerable effect was observed for LNB followed by LNT regarding the turnover, thus