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

Methods: X-ray crystallography was used to determine the structures of BiBga42A in the apo- and liganded forms.
The roles of the amino acid residues that were presumed to be involved in catalysis and substrate recognition were
examined by a mutational study, in which kinetic parameters of each mutant were determined using 4-nitrophenyl-
β-D-galactoside, lacto-N-biose I, LNT, and lacto-N-neotetraose (LNnT) as substrates. Conservation of those amino
acid residues was examined among structure-determined GH42 β-galactosidases.

Results: Crystal structures of the wild-type enzyme complexed with glycerol, the E160A/E318A double mutant
complexed with galactose (Gal), and the E318S mutant complexed with LNT were determined at 1.7, 1.9, and 2.2 Å
resolutions, respectively. The LNT molecule (excluding the Gal moiety at subsite +2) bound to the E318S mutant is
recognized by an extensive hydrogen bond network and several hydrophobic interactions. The non-reducing end
Gal moiety of LNT adopts a slightly distorted conformation and does not overlap well with the Gal molecule bound
to the E160A/E318A mutant. Twelve of the sixteen amino acid residues responsible for LNT recognition and
catalysis in BiBga42A are conserved among all homologs including β-1,6-1,3-galactosidase (BlGal42A) from
Bifidobacterium animalis subsp. lactis.

Conclusion: BlGal42A is active on 3-β-galactobiose similarly to BiBga42A but is inactive on LNT. Interestingly, we
found that the entrance of the catalytic pocket of BlGal42A is narrower than that of BiBga42A and seems not easily
accessible from the solvent side due to the presence of two bulky amino acid side chains. The specificity difference
may reflect the structural difference between the two enzymes.

Keywords: lacto-N-tetraose, glycoside hydrolase family 42, β-galactosidase, bifidobacteria, human milk
oligosaccharides, crystal structure

INTRODUCTION
Human milk not only fulfills the nutritional requirements of newborns but also guides microbiota
[1]
development in the infant gut Human milk oligosaccharides (HMOs), a collective term for sugars with a
degree of polymerization of ≥ 3, are bioactive compounds shown to be instrumental in the formation of
[2-6]
bifidobacteria-rich gut microbiota . HMOs are present at a concentration of 10~20 g/L in breast milk and
are the third most abundant solid material after lactose (Lac) and lipids. HMOs are assumed to reach the
colon intact because they are resistant to pancreatic digestion . A unique feature of HMOs that
[7]
distinguishes them from other mammalian milk oligosaccharides is the richness of type-1 chains (Galβ1-
[8]
3GlcNAc-O-R) . As such, lacto-N-tetraose (LNT, Galβ1-3GlcNAcβ1-3Galβ1-4Glc) is present as the most
[3]
abundant core structure of HMOs [3,8,9] . The type-1 chain is resistant to most bacterial β-galactosidases , and
its hydrolysis requires a specific subgroup of glycoside hydrolase family (GH) 42 enzymes that infant gut-
associated bifidobacterial species possess. We previously reported that Bifidobacterium longum subsp.
infantis (B. infantis) ATCC 15697, which directly internalizes HMOs into its cells using ABC
transporters [3,10] , encodes three intracellular GH42 homologs in its genome. Among the three paralogs, only
a β-galactosidase termed BiBga42A (Blon_2016) hydrolyzes LNT . One of the remaining two GH42
[11]
enzymes, BiBga42B (Blon_2013), was later shown to be specific for β-1,4-galactooligosaccharides . A study
[12]
by James et al. showed that inactivation of lntA, a BiBga42A homolog gene (Bbr_0529), in Bifidobacterium
breve UCC2003 abolishes the ability of the strain to grow on LNT as a sole carbon source, while the
mutation neither affects its ability to utilize Lac nor lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-
4Glc) . The genome of B. breve UCC2003 encodes an additional GH42 homolog , although its specificity
[14]
[13]
has not been determined. GH42 members have thus acquired diversified functions during evolution. Note
that B. bifidum and several strains of B. longum subsp. longum (B. longum) employ an alternative strategy to
degrade LNT. These bifidobacteria extracellularly degrade LNT into lacto-N-biose I (LNB) and lactose (Lac)
by lacto-N-biosidases belonging to GH20 [15,16] (for B. bifidum) and GH136 [5,17,18] (several strains of B. longum)
. The released LNB is then imported by a specific ABC transporter (GltABC) for assimilation [19,20] .