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Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14 Page 3 of 17

As our diet contains different sets of β-galactosides, which are represented by HMOs during breastfeeding
and by galactan oligosaccharides after weaning, precise characterization of GH42 members is of
fundamental importance to obtain a better understanding of how the gut microbiota is shaped and affected
by dietary β-galactosides during the early stages of life.

Since our first report of the crystal structure of a GH42 β-galactosidase from Thermus thermophilus A4 ,
[23]
the three-dimensional structures of 8 members of GH42 have been reported [24-31] . Nonetheless, only a few
GH42 members have been subjected to substrate specificity analysis, and crystal structures of GH42
enzymes are available only in the apo form and/or the product Gal-complexed form. Here, we report the
crystal structures of BiBga42A wild-type (WT) enzyme complexed with glycerol, E160A/E318A mutant
complexed with Gal, and E318S mutant complexed with LNT. This study is the first to show a substrate-
complexed form of GH42 members.

METHODS
Chemicals
4-Nitrophenyl-β-D-galactoside (pNP-Gal) was purchased from Wako Pure Chemical Industries (Osaka,
Japan). LNT and LNnT were provided as gifts from Glycom A/S (Hørsholm, Denmark). LNT was further
[32]
purified by the method described by Ojima et al. . LNB was prepared as described previously . Other
[33]
reagents of analytical grade were obtained from commercial sources.
Site-directed mutagenesis
The QuikChange site-directed mutagenesis method (Agilent Technologies, CA, USA) was used for
introducing amino acid substitutions. pET23b (Merck Millipore, MA, USA) carrying the gene for WT Bi
Bga42A with a C-terminal hexahistidine-tag [Bga42A-(His) ] was used as the template . The following
[11]
6
primers and their complementary strands were used for the mutagenesis (mutation sites are underlined): 5'-
gccagcccggtgccgcacagcactggcgcgcc-3' (for R121A), 5'-catgtgagcaacgcgtacggctgccac-3' (for E160A), 5'-
cgacggcaacgcaatgaacccggg-3' (for F221A), 5'-cagaccaccaacttcggcgtctccgcg-3' (for M262G), 5'- tccaacgaccattt
cttctcgcccggc-3 (for Y287F), 5'-tggttcctcatggcgcattccacgtcc-3' (for E318A), 5'-tggttcctcatgggccattccacgtcc-3'
(for E318G), 5'-tggttcctcatgcagcattccacgtcc-3' (for E318Q), 5'-tggttcctcatgagccattccacgtcc-3' (for E318S), 5'-
ccgccgtcaacgcacgcccgaccaac-3' (for W326A), and 5'-cgtcaactgggcaccgaccaactac-3' (for R327A). After
sequence confirmation, respective plasmids were introduced into the Escherichia coli BL21 (DE3) ΔlacZ
strain for expression .
[11]

Expression and purification of Bga42A variants
The WT Bga42A enzyme from B. infantis ATCC 15697 and its mutant enzymes were expressed and purified
by a previously described method , which involved Ni-nitrilotriacetic acid (NTA) affinity chromatography
[12]
(Qiagen, Hilden, Germany), MonoQ 5/50 GL anion exchange chromatography (GE Healthcare Bio-
Sciences, Uppsala, Sweden), and Superdex 200 10/300 GL size exclusion chromatography (GE Healthcare
Bio-Sciences). When comparing LNT-hydrolyzing activity among E318A, E318G, E318Q, and E318S
mutants, the enzymes were purified by Ni-NTA column chromatography only. The purified enzymes were
concentrated using Amicon Ultra Centrifugal Filters (30 K) (Merck Millipore), dialyzed against 50 mM
sodium phosphate buffer (pH 7.0) containing 0.05% Tween-20, and stored at 4 ºC. The enzymes were stable
for at least one month under the conditions. The protein was quantified using a theoretical absorption
coefficient at 280 nm, calculated based on the sequence (https://web.expasy.org/protparam/).

Enzyme assay
pNP-Gal, LNB, LNT, and LNnT were used as the substrates. The release of Gal was colorimetrically
quantified as described previously [12,34] , in which the released Gal was continuously converted to 6-phospho-