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Page 8 of 17 Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14

Figure 2. Stereoviews of the active site of BiBga42A. (A) E160A/E318A-Gal (cyan) and bound α-Gal (yellow) is superimposed with
WT-GOL (white, thin sticks except for glycerol). W200 and F221 are from the neighboring molecule (light purple); (B) and (C) E318S-
LNT (green) and bound LNT (yellow) focused on LNB (Galβ1-3GlcNAc) disaccharide structure bound in subsites -1 and +1 (B) and Lac
(Galβ1-4Glc) disaccharide structure in subsites +2 and +3 (C). In (B), E160A/E318A-Gal (cyan) and the side chain of Glu-318 in WT-
GOL (white) are superimposed as thin sticks. W200 and F221 are from the neighboring molecule (dark green). BiBga42A: a glycoside
hydrolase family 42 β-galactosidase; LNT: lacto-N-tetraose; WT-GOL: WT enzyme complexed with glycerol.

O
adopts a slightly distorted conformation toward H , and the C6 hydroxymethyl group takes a gg rotamer
5
conformation. The Cremer-Pople parameters (ψ, φ, and Q) of the Gal in subsite -1 are 324.6°, 29.3°, 0.559
[48]
and 323.6°, 28.6°, 0.566 for chains A and B, respectively. The O6 atom of the Gal moiety makes a water-
mediated intramolecular hydrogen bond with the O4 atom of the GlcNAc moiety within the LNT molecule
[Figure 2B]. The conformational change at the O6 atom of the Gal moiety resulted in the loss of two
hydrogen bonds formed with Trp-326 and His-369 in the E160A/318A-Gal [Figure 2A]. Loss of hydrogen
bonds is also found between the O3 atom of the Gal moiety and the side chain of Arg-121 and between the
O2 atom of the Gal moiety and the side chains of Asn-159 and Asp-285. In E318S-LNT, the O3 atom of the
Gal interacts with the side chains of Asp-24 and His-156 via water-meditated hydrogen bonds. The O2 atom
of the Gal in E318S-LNT forms hydrogen bonds with the side chains of Glu-160 and Tyr-287, the latter
interacting with the anomeric O1 atom of the α-Gal in E160A/E318A-Gal [Figure 2A]. The carboxyl group