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[Table 2]. Genome sequence-completed, type strains of 14 species/subsp. were selected for the analysis.
Twelve species/subsp. were found to possess BiBga42A homologs with 62%-96% identity. The lowest hit
(62%) was observed for BlGal42A. Overall identities among the retrieved 12 homologs were between 59%
and 96%, and all 16 residues involved in substrate recognition [Figure 3A] were conserved, as revealed by
ClustalW analysis . Interestingly, the above-mentioned Gly-219 is also conserved among the homologs
[44]
including BlGal42A in the sequence alignment [Figure 4B and Supplementary Figure 3]. Notably, Bl42GalA
has one amino acid insertion in the corresponding loop, which might result in the protrusion of Glu-223 at
the entrance. The twelve homologs possess a glutamic acid or aspartic acid residue in the corresponding
position in the sequences. The multiple alignments also revealed sequence variance at the Gly266-Thr268
and Ala-536 positions among the homologs. Further enzymatic characterization and mutational analysis are
necessary to examine how these homologs efficiently hydrolyze LNT and related tetra- or longer
oligosaccharides.

Concluding remarks
GH42 enzymes, which are found exclusively in microbes, i.e., bacteria, archaea, and fungi, were previously
thought to be involved in the degradation of plant-derived galactooligosaccharides released from
galactans [51,52] . However, a study on BiBga42A, which was first described to be specific for LNT but later
[11]
found to act on a variety of both β-(1→3)- and β-(1→6)-linked galactosides , revealed that GH42 β-
[12]
galactosidases target oligosaccharides of both plant and animal origins. A recent in silico analysis also
demonstrated that the expression of most GH42 β-galactosidase genes is not subject to the regulation by
NagR, a global transcriptional regulator of animal host-glycan degradation genes, in the genus
[53]
Bifidobacterium . In B. infantis, the transcriptional levels of the three GH42 genes were not affected by the
carbon sources used for cultivation, which may represent “enzymatic preparedness in gut microbes” as
mentioned previously .
[54]
Using BiBga42A, a well-characterized GH42 member, our study presents, for the first time, the structure of
a GH42 enzyme complexed with its substrate. Comparison between the two liganded structures showed that
the Gal moiety of LNT occupies the subsite -1 that is sterically different from that observed for the Gal in
E160A/E318A-Gal and takes a different sugar conformation. The result may represent the dynamics in the
catalytic cycle adopted by GH42 enzymes and may explain the lack of transglycosylation activity of anomer-
retaining GH42 members. The donor sugar (Gal) bound at subsite -1 and an acceptor sugar bound at +
subsite(s) may not be arranged suitably for transglycosylation to proceed. Overall, our study warrants
further structural analysis of GH42 members with varied substrates and inhibitors to understand the
catalytic mechanism and the molecular basis of different substrate specificities among the members, which
may help us design novel prebiotics with distinct sugar linkages and compositions.