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Page 6 of 17 Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14

Table 1. The kinetic parameters of BiBga42A variants for pNP-Gal, LNB, LNT, and LNnT
pNP-Gal LNB LNT LNnT
WT K (mM) 0.57 25 2.7 15
m
-1
k (s ) 580 31 86 15
cat
R121A K (mM) 1.4 42 16 35
m
-1
k (s ) 32 0.79 3.6 0.53
cat
E160A K (mM) 0.94 nd b nd nd
m
-1
k (s ) 3.4 nd nd nd
cat
F221Aa K (mM) 1.2 27 8.4 24
m
-1
k (s ) 110 1.4 11 6.4
cat
M262G K (mM) 1.1 14 3.0 13
m
-1
k (s ) 12 0.99 2.9 0.13
cat
Y287F K (mM) 1.0 16 2.6 18
m
-1
k (s ) 6.0 0.18 1.1 0.13
cat
E318A K (mM) 1.4 nd nd nd
m
-1
k (s ) 0.20 nd nd nd
cat
W326A K (mM) 12 33 7.7 64
m
-1
k (s ) 44 0.054 0.14 0.13
cat
R327A K (mM) 0.92 53 11 52
m
-1
k (s ) 380 21 60 11
cat
a b
The amino acid residue from the neighboring subunit in the trimer; not determined. BiBga42A: a glycoside hydrolase family 42 β-galactosidase;
LNB: lacto-N-biose I; LNT: lacto-N-tetraose; LNnT: lacto-N-neotetraose; pNP-Gal: 4-Nitrophenyl-β-D-galactoside; WT: wild-type.
many hydrogen bonds and a stacking interaction. The O6 atom of the sugar forms hydrogen bonds with the
side chain nitrogen atoms of Trp-326 and His-369, while the axial O4 atom is recognized by Arg-121 and
Glu-366. Arg-121 is also involved in the recognition of the O3 atom of the sugar. The O3 atom also forms
water-mediated hydrogen bonds with the Nε1 atom of Trp-200 from the neighboring subunit of the trimer.
Trp-200 is located at the tip of a long helix in domain A [Figure 1]. The O2 atom of the Gal makes hydrogen
bonds with the side chains of Asn-159 and Asp-285. The O2 atom and Asp-285 also form a hydrogen bond
via a water molecule. The water molecule is located at the corresponding position of a side chain oxygen
atom of Glu-318 (nucleophile) observed in WT-GOL. The α-anomeric O1 atom is recognized by the side
chains of Asp-285 and Tyr-287 via hydrogen bonds. The side chain of Phe-356 makes a stacking interaction
with the hydrophobic C4 region of the pyranose ring. All of these residues are conserved within the
members of GH42 β-galactosidases with reported structures [Figure 3A]. In the crystal structure of the β-
galactosidase from Niallia circulans (formerly called Bacillus circulans) subsp. alkalophilus (Bca-β-Gal), the
residue corresponding to Trp-326 of BiBga42A is shifted away .
[26]
As mentioned above, even in the E160A/E318A double mutant, the substrate hydrolysis occurred within 2
days of crystal growth. Crystal soaking experiments were unsuccessful. We then replaced Glu-318 with
glycine (E318G), glutamine (E318Q), or serine (E318S). Using the His-tag affinity-purified preparations at
high concentrations (9 mg/ml), we examined their remaining LNT-hydrolyzing activity. As a result, the
lowest activity was detected for the E318S mutant [Supplementary Figure 2]. An LNT-complexed crystal
was indeed obtained for the mutant, and the structure was determined at 2.2 Å resolution (E318S-LNT) [
Supplementary Table 1]. The asymmetric unit of the E318S-LNT crystal contained two molecules (chains A
and B) of BiBga42A, and they are virtually the same (Cα RMSD = 0.106 Å). Each chain in the asymmetric