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Page 4 of 17 Gotoh et al. Microbiome Res Rep 2023;2:20 https://dx.doi.org/10.20517/mrr.2023.14

glucono-1,5-lactone with the production of an equimolar of Thio-NADH. The reaction mixture contained
varied concentrations of each of the substrates in 100 mM citrate-phosphate buffer (pH 6.5) supplemented
with 10 U/mL hexose mutarotase (FUJIFILM Wako Pure Chemical, Osaka, Japan), 5 U/mL galactokinase,
2.5 U/mL UDP-glucose-hexose-1-phosphate uridylyltransferase, 10 U/mL phosphoglucomutase (Sigma-
Aldrich, MO, USA), 5 U/mL glucose 6-phosphate dehydrogenase (Sigma-Aldrich), 1 mM UDP-Glc, 1 mM
+
ATP, 0.625 mM Thio-NAD (Oriental Yeast, Tokyo, Japan), 0.0125 mM Glc-1,6-bisphosphate, and 12.5
mM MgCl in a total volume of 40 μL. Galactokinase (GalK, BLLJ_0339) and UDP-glucose-hexose-1-
2
phosphate uridylyltransferase (GalT, BLLJ_0398) from B. longum JCM 1217 were prepared as described
previously [33,34] . The mixture was preincubated at 37 ºC for 5 min, to which 10 μL of similarly preincubated
enzyme solution diluted with 50 mM sodium phosphate buffer (pH 6.5) containing 0.05% Tween-20 was
added to initiate the reaction. The reaction was monitored at 37 ºC by measuring the absorbance at 400 nm
(Thio-NADH) every 2 min for 120 min. A Multiskan GO microplate reader (Thermo Fisher Scientific, MA,
USA) was used for spectrophotometry. The kinetic parameters were calculated by curve-fitting the
experimental data with the Michaelis-Menten equation, using KaleidaGraph 4.0 (Synergy Software, Tokyo,
Japan).

Crystallography
The WT enzyme, E160A/E318A double mutant, and E318S mutant were purified as described above. The
WT and E160A/E318A proteins were dialyzed against 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-
yl]ethanesulfonic acid-KOH buffer (pH 7.0) containing 150 mM NaCl, while E318S protein was dialyzed
against 20 mM 3-(N-morpholino)propanesulfonic acid (MES)-NaOH buffer (pH 6.0) containing 0.05%
Tween-20. The hanging drop vapor diffusion method was used for crystallization. The crystal of WT
complexed with glycerol was obtained by mixing 1 μL of a protein solution (20 mg/mL) containing 100 mM
Gal with an equal volume of a reservoir solution consisting of 0.1 M KSCN, 30% PEG MME 2000, and 25%
glycerol (cryoprotectant). The crystal of E160A/E318A complexed with Gal was obtained by mixing 1 μL of
a protein solution (20 mg/mL) containing 60 mM LNT with an equal volume of a reservoir solution
consisting of 0.1 M MES-NaOH buffer (pH 6.5), 0.1 M (CH COO) Mg, 10% PEG 10000, and 25% glycerol
3
2
(cryoprotectant). The crystal of E318S complexed with LNT was obtained by mixing 1 μL of a protein
solution (20 mg/mL) containing 100 mM LNT with an equal volume of a reservoir solution consisting of 5
mM MES-NaOH buffer (pH 5.8), 0.1 M KSCN, 30% PEG MME 2000, and 20% ethylene glycol
(cryoprotectant). The crystals grew at 20 ºC within 2 days in all cases. The crystals were flash-cooled in a
nitrogen stream at 100 K. X-ray diffraction data were collected at 100 K at the beamline BL-5A at the
Photon Factory of the High Energy Accelerator Research Organization (KEK, Tsukuba, Japan, λ = 1.0 Å).
Preliminary diffraction data were collected at other beamlines at Photon Factory and SPring-8 (Hyogo,
Japan). The data sets were processed using XDS (Jan 10, 2022) and Aimless (0.7.9) . Molecular
[36]
[35]
replacement was performed using MOLREP (11.9.02) . Model building and refinement were performed
[37]
using Coot (0.9.8.6) and Refmac (5.8.0403) . The Dali Server was used for structural comparison .
[39]
[38]
[40]
Molecular interface analysis was performed using the PDBePISA server . Molecular graphic images were
[41]
prepared using PyMOL (2.5.4) (Schrödinger, NY, USA). Stereographic figures were created using the “ray
angle =” command, specifying -3 and +3 degrees for the left and right panels, respectively.
Phylogenetic tree construction
[31]
The structure-determined β-galactosidases [23-30] and α-arabinopyranosidase listed in GH42 of the
Carbohydrate-Active enZYmes database and BiBga42A were used for the tree construction. A β-
[42]
galactosidase from Bifidobacterium adolescentis was omitted from the analysis because the structure does
not contain the catalytic domain (Midwest Center for Structural Genomics). The maximum likelihood tree
was constructed using MegaX, based on the sequences aligned using the ClustalW algorithm with default
settings .
[43]