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Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14 Page 5 of 17

Search for BiBga42A homologs among several bifidobacterial species
The occurrence of BiBga42A homologs among several Bifidobacterium species was examined using the
Tblastn program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Genome sequence-completed, type strains of B.
adolescentis ATCC 15703, Bifidobacterium angulatum JCM 7096, Bifidobacterium animalis subsp. animalis
ATCC 25527, B. animalis subsp. lactis (B. lactis) DSM 10140, B. bifidum JCM 1255, B. breve JCM 1192,
Bifidobacterium catenulatum subsp. catenulatum JCM 1194, B. catenulatum subsp. kashiwanohense JCM
15439, Bifidobacterium dentium JCM 1195, Bifidobacterium eulemuris DSM 100216, Bifidobacterium
lemurum DSM 28807, B. longum JCM 1217, Bifidobacterium pseudocatenulatum JCM 1200, and
Bifidobacterium pseudolongum subsp. globosum DSM 20092 were used for the homolog search. Alignment
[44]
of the retrieved homologs was carried out using the ClustalW program with default settings .

RESULTS AND DISCUSSION
Crystal structures of BiBga42A
First, we determined the structure of the WT enzyme complexed with glycerol (WT-GOL) at 1.7 Å
resolution [Supplementary Table 1]. The electron density of a glycerol molecule, which was used as a
cryoprotectant, was observed at the active site, even though the crystals were grown in the presence of Gal [
Supplementary Figure 1A]. BiBga42A has a three-dimensional structure similar to other GH42 enzymes that
have three domains consisting of a (β/α) barrel (domain A), an α/β fold (domain B), and an anti-parallel β-
8
sandwich (domain C) [Figure 1A]. A Dali structural search revealed that BiBga42A is most similar to
[45]
GH42 β-1,6-galactosidase from B. bifidum S17 (BbgII, PDB ID: 4UCF) with the root-mean-square
[24]
deviation (RMSD) = 0.6 Å for 684 Cα atoms (Z score = 56.6 and sequence identity = 76%). The second hit
was GH42 β-1,6-1,3-galactosidase from B. lactis Bl-04 (BlGal42A, PDB ID: 4UOZ) with RMSD = 0.6 Å for
[25]
685 Cα atoms (Z score = 55.9 and sequence identity = 62%). The asymmetric unit of the WT-GOL crystal
contained one molecule of BiBga42A, and it forms a homotrimer with symmetry-related molecules by a
crystallographic 3-fold axis [Figure 1B]. A PISA molecular interface analysis calculated that approximately
27% of the entire surface area of the trimer (17,082 Å of 64,278 Å) was buried. Our previous study using
calibrated gel filtration chromatography also suggested that BiBga42A forms a trimer in solution . The
[11]
trimeric structure of BiBga42A resembles a flowerpot, like other GH42 enzymes [23-25] .

Glu-160 and Glu-318 in BiBga42A were predicted to be the acid/base catalyst and the nucleophile,
respectively, based on previous studies of GH42 enzymes and sequence alignment [46,47] . The k values of the
cat
respective alanine mutants (E160A and E318A) towards pNP-Gal dropped by 170- and 2,900-fold compared
to the WT enzyme, respectively, without affecting K values (within 2.5-fold change) [Table 1 and
m
Supplementary Figure 2]. Based on these results, we first attempted co-crystallization of E318A with LNT.
However, in a preliminary experiment, we found that the purified mutant can hydrolyze LNT when present
at a high concentration (9 mg/mL) [Supplementary Figure 2]. Therefore, we also replaced the acid/base
catalyst Glu-160 with alanine and performed co-crystallization with LNT using the preparation (E160A/
E318A double mutant). The crystals obtained within 2 days were used for X-ray diffraction data collection.
However, LNT was also hydrolyzed during the crystallization under these conditions, because only the
electron density of the product Gal in the α-configuration (α-Gal) was observed in subsite -1 of the active
site [Supplementary Figure 1B]. The Gal-complexed structure was determined at 1.9 Å resolution (E160A/
E318A-Gal) [Supplementary Table 1]. α-Gal was also observed in the crystal structures of several other
GH42 enzymes [23-26] . The asymmetric unit contained six molecules (two trimers) of BiBga42A. The main
chain structures of the six polypeptides (chains A-F) were virtually the same (Cα RMSD < 0.107 Å for all
chain pairs), and an α-Gal molecule was similarly bound in each chain. Therefore, we mainly describe the
4
chain A molecule. The pyranose ring of the α-Gal adopts an undistorted C conformation, and the C6
1
hydroxyethyl group takes a gt conformation [Figure 2A and Supplementary Figure 1B]. The glycerol
molecule in WT-GOL occupies the C2-C4 position of the Gal. The Gal recognition by the protein involves