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Page 12 of 17 Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14

Figure 4. Comparison of the active site structures between BiBga42A and BlGal42A. (A) Superimposition of the active sites of Bi
Bga42A (E318S-LNT, green for protein and yellow for LNT) and BlGal42A (orange for protein and Gal); (B) Molecular surface of Bi
Bga42A (left) and BlGal42A (right). Right, LNT in BiBga42A is superimposed as thin sticks. Surface areas of the residues contributing to
shaping the different entrance sizes are colored green or orange.BiBga42A: a glycoside hydrolase family 42 β-galactosidase; LNT: lacto-
N-tetraose; BlGal42A: β-1,6-1,3-galactosidase.

except for Glu-366, which is displaced upon LNT binding [Figure 4A]. However, the main chain trace and
the side chain conformation of Ser-272-Asn-274 in BlGal42A are different from those of Gly266-Thr268 in
BiBga42A. Upon examination of the surface models of the two structures, we found that the catalytic pocket
of BlGal42A is narrower than that of BiBga42A [Figure 4B]. The side chain of Ser-272 makes the pocket of
BlGal42A slightly tighter. The protruding side chain of Glu-223 located on a loop of BlGal42A especially
occludes the entrance of the pocket. Phe-542, which is located in the loop extending from the other side,
also covers the entrance of the catalytic pocket of BlGal42A. BiBga42A substitutes Gly-219 and Ala-536 for
Glu-223 and Phe-542 of BlGal42A, respectively. These bulky residues might interfere with the efficient
access of longer oligosaccharide substrates to the catalytic site of BlGal42A.

We also examined the conservation of these amino acid residues among the BiBga42A homologs of several
Bifidobacterium species that reside in the gastrointestinal tract (GIT) of humans and non-human mammals