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Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14 Page 9 of 17

Figure 3. Sequence comparison within the structure-determined GH42 members. (A) The amino acid residues involved in ligand
recognition in BiBga42A were aligned with eight structure-determined GH42 β-galactosidases using the ClustalW program [44] . The
residue numbers are based on BiBga42A. The sugar moieties interacting with the respective amino acid residues are indicated by their
subsite positions. Asterisks indicate the residues from the neighboring subunit; (B) Structure-determined enzymes, i.e., eight β-
galactosidases (blue) and one α-arabinopyranosidase (brown), and BiBga42A (bold black) were used for the tree construction. The
Uniprot numbers, PDB IDs, and taxonomic names are shown. β-Galactosidase from B. adolescentis (PDB ID: 5VYM) was omitted from
the analysis because the structure does not contain the catalytic domain. The maximum likelihood tree was constructed using MegaX
[43]
based on the sequences aligned using ClustalW with default settings . The lowest amino acid identity detected among the members is
20% between B. lactis A0A2H4A2Z3 and B. bifidum E3EPA1, G. stearothermophilus F8TRX0, or Thermus sp. O69315, while the highest
[24]
one is 76% between B. infantis B7GUD7 (BiBga42A) and B. bifidum E3EPA1 (BbgII). The sequences are: B. bifidum E3EPA1 , B. animalis
[25] [26] [27]
subsp. lactis C6A6W5 , N. circulans subsp. alkalophilus A0A4D6T0U7 , G. stearothermophilus F8TRX0 , Rahnella sp.
[28] [29] [30] [23]
A0A0B4U8I5 , H. lacusprofundi B9LW28 , Marinomonas sp. A0A6P3CWF7 , T. thermophilus O69315 , and B. animalis subsp.
lactis A0A2H4A2Z3 [31] . BiBga42A: a glycoside hydrolase family 42 β-galactosidase; GH: glycoside hydrolase.
of Glu-160 is suitably poised for proton donation to the leaving group as it is located 3.3 Å from the
glycosidic bond oxygen of the LNB (Galβ1-3GlcNAc) unit in LNT. The stacking interaction between the
phenolic ring of Phe-356 and the C4 region of the Gal remains unchanged. As the Gal-recognition modes
between E160A/E318A-Gal and E318S-LNT differ, the two Gal do not overlap well with each other even at
the C1 atom [Figure 2B], to which the nucleophile Glu-318 residue is thought to attack to form a galactosyl-
[47]
enzyme intermediate during catalysis . The C1 atom of the Gal moiety at subsite -1 in E318S-LNT has
moved away from the side chain of Ser-318 with a distance of 6.2 Å. In addition, the C1 atom was located 4.3
Å from the nearest side chain oxygen atom (Oε1) of the superimposed Glu-318 of WT-GOL, and the
configuration was not suitable for the in-line nucleophilic attack to the anomeric carbon. These
observations might suggest that LNT was bound in a catalytically incompetent mode in the structure. The