Function and Structure of Lacticaseibacillus casei GH35 β-Galactosidase LBCZ_0230 with High Hydrolytic Activity to Lacto-N-biose I and Galacto-N-biose

Abstract

β-Galactosidase (EC 3.2.1.23) hydrolyzes β-D-galactosidic linkages at the non-reducing end of substrates to produce β-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative β-galactosidase belonging to glycoside hydrolase family 35 (GH35). This enzyme is encoded by the gene included in the gene cluster for utilization of lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-β-galactosidase (EC 3.2.1.85). However, its 6-phospho-β-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. casei JCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recombinant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl β-D-galactopyranoside, p-nitrophenyl β-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-β-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite −1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae β-galactosidase BgaC and not suitable for binding to 6-phospho-β-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a β-galactosidase. According to the prediction of LNB's binding mode, aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glucosamine residue of LNB at subsite +1.

Abbreviations

GalNAc, N-acetyl-D-galactosamine; GH, glycoside hydrolase; GH35, GH family 35; GlcNAc, N-acetyl-D-glucosamine; GNB, galacto-N-biose; LNB, lacto-N-biose I; oNPGal, o-nitrophenyl β-D-galactopyranoside; oNPGal6P, o-nitrophenyl 6-phospho-β-D-galactopyranoside; pNPGal, p-nitrophenyl β-D-galactopyranoside; PTS, phosphoenolpyruvate: sugar phosphotransferase system; RMSD, root mean square deviation.

INTRODUCTION

β-Galactosidase (EC 3.2.1.23) is a retaining glycoside hydrolase that hydrolyzes the β-D-galactosidic linkage at the non-reducing end of substrates. It is known that a variety of enzymes hydrolyze various β-D-galactosides, such as lactose (Galβ1-4Glc) and lacto-N-biose I (LNB; Galβ1-3GlcNAc). The enzymes highly active to lactose are industrially utilized to produce galactooligosaccharides through transgalactosylation and degrade lactose in milk for the production of lactose-free dairy products.¹⁾²⁾ Acid tolerant β-galactosidases are helpful as a digestive supplement to alleviate symptoms of lactose intolerance.³⁾⁴⁾ According to the sequence-based classification of glycoside hydrolases,⁵⁾ β-galactosidase is classified into glycoside hydrolase (GH) families 1, 2, 3, 5, 16, 35, 42, 50, 147, 165, and 173. A recent functional genomics study discovered β-galactosidases categorized into new GH families.⁶⁾⁷⁾⁸⁾

GH family 35 (GH35) β-galactosidases are distributed in various organisms, including mammals, plants, and microorganisms. In the lysosomes of mammalian tissues, acid β-galactosidase belonging to this family cleaves the β-D-galactosidic linkage of gangliosides, glycoproteins, and glycosaminoglycans.⁹⁾ Plant β-galactosidases are found in germination seeds and ripening fruits.¹⁰⁾¹¹⁾ Several plant enzymes that release D-galactose from the sidechains of xyloglucan, pectin, and arabinogalactan proteins have been identified.^12)13)14)15) Bacteria and fungi, which utilize plant polysaccharides as carbon sources, also possess β-galactosidase with similar substrate specificities.^16)17)18)19) It has been found that bacteria habiting in mammalian bodies, such as Streptococcus pneumoniae and Akkermansia muciniphila, have GH35 enzymes specific to β-(1→3)-D-galactopyranosides that link to N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc).^20)21)22) These β-D-galactoside structures are included in the type I human milk oligosaccharides, mucin glycans, and glycan bound to blood proteins such as fetuin.^23)24)25)

Lacticaseibacillus casei, which was recently renamed from Lactobacillus casei,²⁶⁾ is one of the most widely studied and utilized probiotic species of lactobacilli.²⁷⁾ The genome of L. casei BL23 contains the gene GnbG, which encodes the GH35 protein. This gene is included in a gene cluster for the utilization of LNB and galacto-N-biose (GNB; Galβ1-3GalNAc) via a phosphoenolpyruvate: sugar phosphotransferase system (PTS).²⁸⁾ Contrary to Bifidobacterium, which imports LNB via an ATP binding cassette transporter and phosphorolyzes it with GH112 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (EC 2.4.1.211),²⁵⁾²⁹⁾ this bacterium lacks both of them. Bidart et al. mentioned that GnbG is a phospho-β-galactosidase (EC 3.2.1.85) that degrades phosphorylated LNB and GNB.²⁸⁾ However, its activity as a phospho-β-galactosidase has not been examined. As reported, GnbG is capable of hydrolyzing o-nitrophenyl β-D-galactopyranoside (oNPGal), LNB, and GNB. GnbG transfers D-galactosyl residue from oNPGal to GlcNAc and GalNAc to produce LNB and GNB, respectively.²⁸⁾³⁰⁾ According to this, GnbG appears to possess the functions of β-galactosidase.

The L. casei JCM1134 strain (same as ATCC393) contains the same gene cluster for LNB/GNB utilization as L. casei BL23 (Fig. 1). LBCZ_0230 from L. casei JCM1134 is homologous to GnbG from L. casei BL23 (sequence identity between LBCZ_0230 and GnbG is 86 %). Therefore, this study investigates the enzymatic characteristics and crystal structure of LBCZ_0230.

Fig. 1. Gene organization for LNB utilization in L. casei BL23 and L. casei JCM1134.

　Black, green, yellow, magenta, and cyan arrows indicate the gene encoding transcriptional regulator, D-galactose-6-phosphate isomerase, N-acetylglucosamine-6-phosphate deacetylase, GH35 enzyme, and component of PTS system, respectively. Gene names of L. casei JCM1134 are indicated by the last three numbers (xxx) of the locus tag (LBCZ_0xxx).

MATERIALS AND METHODS

Construction of expression plasmid of LBCZ_0230. PCR amplified the LBCZ_0230 gene (GenBank ID, BAN73398.1) from the genomic DNA of L. casei JCM1134 (Japanese Collection of Microorganisms, Tsukuba, Japan) utilizing KOD FX Neo DNA polymerase (Toyobo Co., Ltd., Osaka, Japan). The following primers were used: 5′-ATGACGACTTTTTCGATCGAGC-3′ (sense orientation) and 5′-CTACTCCTCCTCATTATTTGG-3′ (antisense orientation). A second PCR was conducted using an amplified DNA fragment as a template. In this experiment, the primers used were 5′-AAGAAGGAGATATACATATGACGACTTTTTCGATCGAGC-3′ (sense orientation) and 5′-CAGTGGTGGTGGTGGTGGTGCTCCTCCTCATTATTTGGTTC-3′ (antisense orientation). Using the In-Fusion HD Cloning Kit (Takara Bio Inc., Kusatsu, Japan), the DNA fragment amplified by the second PCR was inserted into pET-23a (Novagen /Merck KGaA, Darmstadt, Germany). In order to verify the sequence of inserted DNA and flanking regions of cloned plasmid, an Applied Biosystems 3130 Genetic Analyzer was employed (Applied Biosystems, Inc., Foster City, CA, USA).

Preparation of recombinant LBCZ_0230. Transformants of E. coli BL21 (DE3) harboring the expression plasmid were grown in 1 L of LB liquid medium supplemented with 100 μg/mL ampicillin until A₆₀₀ reached 0.5. An induction culture was performed at 18 °C for 20 h in the presence of 0.1 mM isopropyl β-D-1-thiogalactopyranoside. The bacterial cells isolated from the culture broth by centrifugation (6,800 ×g, 4 °C, 10 min) were resuspended in 20 mM imidazole-HCl buffer (pH 7.0) containing 0.5 M NaCl and disrupted by sonication. Centrifugation was used to obtain the cell-free extract. The recombinant enzyme was purified using a Ni²⁺-immobilized affinity column chromatography utilizing Chelating Sepharose Fast Flow (2.6 cm I.D. × 3 cm; GE Healthcare, Uppsala, Sweden). After washing the column with a 20 mM imidazole-HCl buffer (pH 7.0) containing 0.5 M NaCl, the adsorption protein was eluted by a linear gradient of imidazole (20-500 mM; elution volume, 100 mL). The pooled fractions were dialyzed against 10 mM sodium phosphate buffer (pH 7.0). Prior to crystallization, LBCZ_0230 was purified with Toyopearl DEAE 650 M column chromatography in 10 mM MES-NaOH buffer (pH 7.0) (2.6 cm I.D. × 15 cm; Tosoh Corporation, Tokyo, Japan). A linear gradient of 0-0.5 M NaCl was used to elute the adsorbate protein (elution volume, 400 mL). Following hydrolysis of the sample in 6 M HCl at 110 °C for 24 h, the amino acid analysis was used to determine the protein concentration of the purified enzyme.

Enzyme activity assay. As a standard assay of LBCZ_0230, the hydrolyzing activity to p-nitrophenyl β-D-galactopyranoside (pNPGal) was measured. A reaction mixture (50 μL) containing an appropriate concentration of the enzyme, 80 mM sodium acetate buffer (pH 4.0), and 2 mM pNPGal (Nacalai Tesque, Inc., Kyoto, Japan), was incubated at 37 °C for 10 min and liberated p-nitrophenol was quantified based on A₄₀₀ after adding 100 μL of 1 M Na₂CO₃ to terminate the reaction. Under these conditions, 1 U of enzyme produces 1 μmol of p-nitrophenol in 1 min.

Effects of pH and temperature on activity and stability. We determined the optimal pH for LBCZ_0230 by analyzing the activity at various pH levels. The pH of the reaction was altered by using 80 mM Britton Robinson buffer (pH 2.5-10.0) as the reaction buffer. In order to determine a stable range of pH and temperature, residual activity was examined after pH and temperature treatments, respectively. The pH treatment was carried out by incubating LBCZ_0230 at 4 °C for 24 h in 20 mM Britton Robinson buffer (pH 2.5-11.5). For the temperature treatment, the enzyme was incubated at 30-60 °C for 20 min in 133 mM sodium acetate buffer (pH 4.0).

Kinetic analysis. We determined the reaction rates of LBCZ_0230 to pNPGal, oNPGal (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), LNB,³¹⁾ GNB,³²⁾ lactose (Nacalai Tesque), allolactose (Galβ1-6Glc; Megazyme International Ireland, Bray, Ireland), and o-nitrophenyl 6-phospho-β-D-galactopyranoside (oNPGal6P; cyclohexylamine salt; Toronto Research Chemicals, Inc., Toronto, ON, Canada). As described above, the enzyme reaction was conducted under the same conditions as the enzyme activity assay. The reaction with substrates other than pNPGal, oNPGal, and oNPGal6P was terminated by heating the sample at 80 °C for 3 min. A₄₀₀ was measured to quantify o-nitrophenol released in the reaction with oNPGal and oNPGal6P. In the reaction with lactose, D-glucose was quantified using the Glucose C-II Test (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and D-galactose was measured using the L-Arabinose/D-Galactose Assay Kit (Megazyme) for the reactions with LNB, GNB, and allolactose. The kinetic parameters of the Michaelis-Menten equation were computed based on the reaction rates at various substrate concentrations through non-linear regression using Grafit ver. 7.0.2 software (Erithacus Software Limited, East Grinstead, UK). In order to determine kinetic parameters, the concentrations of pNPGal, oNPGal, LNB/GNB, and lactose were 0.0125-0.5, 0.05-1, 0.1-2, and 10-160 mM, respectively. The substrate concentrations for measuring reaction velocity with allolactose and oNPGal6P were 2 and 1 mM, respectively.

Blue native PAGE. An analysis of blue native PAGE³³⁾ was performed under nondenaturing conditions to determine the molecular mass of LBCZ_0230. The analytical sample was prepared using a Native PAGE Sample Prep Kit (Life Technologies Corporation, Carlsbad, CA, USA). Electrophoresis was conducted on Native PAGE Novex 4-16 % Bis-Tris Gels (Life Technologies) at 150 V for 115 min on ice. Molecular mass was calibrated using Native Mark Unstained Standard (Life Technologies).

Crystallization and data collection. Initial crystallization conditions were screened using crystallization kits from Hampton Research (Journey Aliso Viejo, CA, USA) by using the hanging drop vapor-diffusion method. First, 1 μL of protein solution (14.1 mg/mL) in 10 mM HEPES-NaOH buffer (pH 7.0) was mixed with an equal volume of reservoir solution. LBCZ_0230 crystals were obtained within two weeks at 20 °C with reservoir solution containing 0.1 M HEPES-NaOH buffer (pH 7.5), 10 % (v/v) 2-propanol, and 200 g/L polyethylene glycol 4000. In X-ray diffraction experiments, the crystals were directly picked up from the crystallization solution and flash-cooled. LBCZ_0230 diffraction data were collected at SPring-8 (Hyogo, Japan) on beamline BL44XU. Dectris Eiger X 16M (Baden, Switzerland) was used as the detector. The XDS program suite was used to index, integrate, scale, and merge the data sets.³⁴⁾ It was assumed that the asymmetric unit of LBCZ_0230 contained four molecules, giving a Matthews coefficient³⁵⁾ and solvent content of 2.24 ųDa^-1 and 45.2 %, respectively. Table 1 summarizes all data collection statistics.

Table 1. Summary of crystallization conditions, data collection, and refinement statistics.

	LBCZ_0230
PDB ID	8H25
Data collection
Beamline	SPring-8 BL44XU
Space group	P21
Unit cell parameters
a, b, c (Å)	92.4, 111.0, 120.3
β (°)	90.02
Wavelength (Å)	0.9000
Resolution range (Å)	47.6-2.29 (2.43-2.29)
Total No. of reflections	409,837 (66,327)
No. of unique reflections	108,220 (17,244)
Rmeas (%)*	8.5 (68.4)
<I/σ (I)>	13.2 (2.38)
CC1/2	0.998 (0.768)
Completeness (%)	99.7 (98.9)
Redundancy	3.79 (3.85)
Refinement
Rwork/Rfree (%)**	19.5/23.2
Twin operator	−h, −k, l
Twin fraction (%)	48.8
No. of atoms
Macromolecules	19,024
Ligand	108
Water	368
B-factors (Å2)
Macromolecules	46.0
Ligand	57.4
Water	35.0
RMSD from ideal
Bond lengths (Å)	0.003
Bond angles (°)	0.886
Ramachandran plot
Favored (%)	96.14
Allowed (%)	3.52
Outliers (%)	0.34

Values in parentheses are for the highest resolution shell.

^*R_meas = Σ_hkl {N(hkl) / [N(hkl) - 1]}^1/2 Σ_i | I_i(hkl) - <I(hkl)> | / Σ_hkl Σ_i I_i(hkl), where <I(hkl)> and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively.

^**R_work = Σ_hkl ||F_obs| - |F_calc|| / Σ_hkl |F_obs|, R_free was calculated for 5 % randomly selected test sets that were not used in the refinement.

Structure solution and refinement. The structure of LBCZ_0230 was determined using the molecular replacement method and the program AutoMR in the PHENIX program package.³⁶⁾³⁷⁾ The structure of S. pneumoniae β-galactosidase BgaC (PDB entry, 4E8D)³⁸⁾ was used as the search model. The refinement process was carried out using the program phenix. refine in conjunction with interactive fitting and rebuilding based on 2F_o−F_c and F_o−F_c electron densities using COOT.³⁵⁾³⁹⁾ Water molecules were constructed based on electron densities. The crystal was pseudomerohedrally twinned with the twin operator (−h, −k, l). As a final step, the structure of LBCZ_0230 was refined using Refmac5's intensity-based twin refinement method.⁴⁰⁾ The final refinement statistics, as well as the Ramachandran analysis by Molprobity⁴¹⁾ are shown in Table 1. The atomic coordinates and structure factors were deposited in the Protein Data Bank (http://www.wwpdb.org/; code 8H25). The structure figures were generated using PyMOL ver. 2.5.0 (Schrödinger, LLC, New York, NY, USA).

RESULTS

Preparation and basic properties of recombinant LBCZ_0230.

The purification of recombinant LBCZ_0230, in which 6 His residues were attached to the C-terminal residue of the protein, from cell-free extracts of E. coli transformants expressing the LBCZ_0230 gene was accomplished by Ni²⁺-immobilized affinity column chromatography. One litter of culture broth yielded 15.3 mg of purified enzyme. It was found that the purified enzyme had a specific activity of 8.67 U/mg in the presence of 2 mM pNPGal at pH 4.0 and 37 °C. The molecular mass of LBCZ_0230 has been estimated to be 63 and 115 kDa, respectively, based on SDS-PAGE and blue native PAGE analyses (Fig. 2). The molecular mass determined by SDS-PAGE was similar to the theoretical mass calculated from the amino acid sequence, 68.7 kDa. Based on these results, it appears that LBCZ_0230 is a dimeric protein under non-denaturing conditions. The highest activity of LBCZ_0230 was observed at pH 4.0. The activity was high (≥70 % of the highest activity) in a broad pH range: pH 3.5-7.1 (Fig. 3). LBCZ_0230 retained ≥90 % of its original activity at pH 3.5-8.0 (4 °C, 24 h) and at ≤50 °C (pH 4.0, 20 min) (Fig. 3). Under acidic conditions, this enzyme is highly active and stable.

Fig. 2. SDS-PAGE and blue-native PAGE analysis of purified LBCZ_0230.

　Lanes M and S are molecular size markers and purified samples, respectively. (A) SDS-PAGE. (B) Blue-native PAGE. LBCZ_0230 showed a single band of 63 and 115 kDa, in SDS-PAGE and Blue-native PAGE, respectively.

Fig. 3. Effect of pH and temperature on the activity and stability of LBCZ_0230.

　(A) Indicates the effect of pH on the relative activity. Closed and open circles indicate activity and stability, respectively. Stability was evaluated by residual activity after incubation at various pH values at 4 °C for 24 h. (B) Temperature stability. Residual activity after incubation at various temperatures for 20 min was measured. Values and error bars are the average and standard deviation of triplicated data, respectively.

Substrate specificity.

In addition to pNPGal, LBCZ_0230 efficiently hydrolyzed oNPGal, LNB, and GNB and exhibited slight hydrolytic activity towards lactose and allolactose. The k_cat/K_m values for pNPGal, oNPGal, LNB, and GNB were 518, 297, 45.4, and 14.4 s^-1mM^-1, respectively (Table 2). Since k_cat values for these substrates were similar, variation in k_cat/K_m was primarily due to changes in K_m values (0.0209-0.707 mM). The k_cat/K_m for lactose (0.00346 s^-1mM^-1) was 4.16 × 10³-150 × 10³ times lower than for these substrates (Table 2). The reaction rate for 2 mM allolactose was 0.0924 ± 0.0058 s^-1. The enzyme did not exhibit detectable activity toward oNPGal6P (< 0.0037 s^-1).

Table 2. Kinetic parameters of LBCZ_0230 for various β-D-galactopyranoside.

Substrate	kcat (s-1)	Km (mM)	kcat/Km (s-1mM-1)
pNPGal	10.8 ± 0.1	0.0209 ± 0.0013	518
oNPGal	14.9 ± 0.1	0.0501 ± 0.0023	297
LNB	10.8 ± 0.1	0.238 ± 0.005	45.4
GNB	10.2 ± 0.1	0.707 ± 0.018	14.4
Lactose	0.695 ± 0.040	201 ± 14	0.00346

Values are shown as average ± standard deviation for values from three independent experiments.

Three-dimensional structure of LBCZ_0230.

Crystal structure of LBCZ_0230 was determined at 2.29 Å resolution (Table 1). Four monomers of LBCZ_0230, which are similar to each other (root mean square deviation (RMSD) of these monomers was 0.06-0.158 Å), were located in an asymmetric unit. Based on molecular assembly predictions using PDBePISA v1.52,⁴²⁾ chains A and C, and B and D form stable dimers in solution (Fig. 4A). This prediction is in agreement with the results of the blue native PAGE analysis described above (Fig. 2). The monomer of LBCZ_0230 is formed by three domains: (β/α)₈-barrel catalytic domain and two β-domains, following the catalytic domain (Fig. 4A). A similarity search using Dali server⁴³⁾ revealed that the three-dimensional structure of LBCZ_0230 is similar to S. pneumoniae BgaC (PDB entry, 4E8D; RMSD, 0.8 Å),³⁸⁾ Bacillus circulans β-galactosidase BgaC (PDB entry, 4MAD; RMSD, 1.5 Å),⁴⁴⁾ human β-galactosidase BgaC (PDB entry, 3THD; RMSD, 1.8 Å),⁴⁵⁾ and so on. The chains A and C, and B and D of LBCZ_0230 overlap well with the dimeric structure of S. pneumoniae BgaC.

Fig. 4. Structural analysis of LBCZ_0230.

　(A) dimeric structure of LBCZ_0230. Chains A and C are shown in an asymmetric unit. Schematic diagram of LBCZ_0230 is shown below the figure. The regions of the catalytic domain, β-domain 1, and β-domain 2 are indicated by black, light gray, and dark gray, respectively. The β-domains 1 is formed by two separated regions. (B) Stereo view of the predicted structure of LBCZ_0230 in complex with D-galactose. LBCZ_0230 (green sticks and gray surface) is superimposed onto S. pneumoniae BgaC in complex with D-galactose (magenta sticks; PDB entry, 4E8C). D-Galactose is represented as yellow sticks. (C) Prediction of LNB binding structure of LBCZ_0230. Stereo view of superimposed structures of LBCZ_0230 (green sticks) and Aspergillus niger β-galactosidase E298Q in complex with Galβ1-3Glc (PDB entry, 5IFT; purple sticks)⁴⁶⁾ is shown. D-Galactosyl and D-glucose residues of Galβ1-3Glc are shown with yellow and orange sticks, respectively. An ideal form of GlcNAc (PDB entry, NAG; cyan sticks) is overlapped on the D-glucose residue of Galβ1-3Glc to predict the substrate binding mode in subsite +1. (D) Stereo view of the predicted structure of LBCZ_0230 in complex with LNB (green sticks and gray surface). Structure of S. pneumoniae BgaC (magenta sticks) is superimposed onto LBCZ_0230. D-Galactosyl and GlcNAc residues of the predicted LNB are shown in yellow and cyan sticks, respectively.

A comparison was made between the substrate binding site of LBCZ_0230 and that of S. pneumoniae BgaC in which D-galactose binds to subsite −1. The superimposition of LBCZ_0230 onto the D-galactose complex of S. pneumoniae BgaC (PDB entry, 4E8C)³⁸⁾ demonstrates that these enzymes have very similar D-galactose binding sites (Fig. 4B). Although the orientation of Trp243 and Tyr455 of S. pneumoniae BgaC in apo-form (PDB entry, 4E8D) differs from that of the D-galactose complex (PDB entry, 4E8C),³⁸⁾ the corresponding residues of LBCZ_0230 (Trp243 and Tyr458, respectively) exhibited similar orientations to those of the D-galactose complex of S. pneumoniae BgaC. Glu156 and Glu238 of LBCZ_0230 are located at the same position of the general acid/base catalyst and catalytic nucleophile of S. pneumoniae BgaC, respectively. All hydroxy groups in D-galactose are capable of forming hydrogen bonds with the surrounding amino acid residues. Glu98 and Tyr305 are predicted to form hydrogen bonds with 6-OH of D-galactose, and there is no space to accommodate the 6-phosphate group of 6-phospho-β-D-galactopyranosides. Thus, consistent with the result of the substrate specificity analysis, the structure of subsite −1 of LBCZ_0230 is unsuitable for hydrolysis of 6-phospho-β-D-galactopyranosides.

To predict the binding mode of LNB to LBCZ_0230, LBCZ_0230 was superimposed onto Aspergillus niger β-galactosidase E298Q in complex with Galβ1-3Glc (PDB entry, 5IFT),⁴⁶⁾ and GlcNAc in ideal form (PDB entry, NAG) was overlapped onto the D-glucose residue of Galβ1-3Glc (Fig. 4C). Most residues at subsite −1 and the catalytic residues of LBCZ_0230 were spatially conserved well with those of A. niger β-galactosidase as shown in the structural comparison between LBCZ_0230 and S. pneumoniae BgaC. Four atoms, 2-C, 3-C, 3-O, and 4-C, of GlcNAc were overlapped well onto corresponding atoms of the D-glucose residue, which takes B_3,O conformation, of Galβ1-3Glc. Based on the predicted binding mode of GlcNAc at subsite +1 of LBCZ_0230 (Figs. 4C and D), aromatic residues, Trp190 (loop 5, connecting fifth β-strand and α-helix of the catalytic domain); Trp240, Trp243, Phe244 (loop 7, connecting seventh β-strand and α-helix of the catalytic domain), and Tyr458 (β5→β6 loop, connecting fifth and sixth β-strands of β-domain 1), are predicted to have hydrophobic interaction with the GlcNAc residue in subsite +1.

DISCUSSION

L. casei is widely used as a probiotic species of lactobacilli,²⁷⁾ and is known to utilize LNB and GNB as carbon sources.²⁸⁾ Bidart et al. postulated that L. casei BL23 metabolizes LNB and GNB via the PTS pathway, and the GH35 protein, GnbG, intracellularly hydrolyzes phosphorylated LNB and GNB as a phospho-β-galactosidase.²⁸⁾ However, the amino acid sequence of GnbG is highly similar to GH35 β-galactosidases, and the phospho-β-galactosidase activity of GnbG has not been examined yet. In this study, a protein from L. casei JCM1134 GH35, LBCZ_0230, which is highly similar to GnbG from L. casei BL23, has been biochemically and structurally characterized. A recombinant LBCZ_0230 produced in E. coli displayed high hydrolytic activity towards β-D-galactosides including pNPGal, oNPGal, LNB, and GNB but not against oNPGal6P. LBCZ_0230 has also been structurally analyzed, which indicates that while subsite −1 of this enzyme is suitable to bind D-galactoside, there is insufficient space within the subsite to accommodate the 6-phospho-D-galactosyl group (Fig. 4B). The functional and structural observations indicate that LBCZ_0230 is not phospho-β-galactosidase but a true β-galactosidase. As L. casei BL23, the LBCZ_0230 gene is part of the gene cluster for the metabolism of LNB and GNB through the PTS pathway (Fig. 1). Because LNB, GNB and GlcNAc are transported to the cytosol by the same PTS, encoded by the genes neighboring the gnbG gene in L. casei BL23,²⁸⁾ and L. casei JCM1134 also possesses the corresponding PTS as shown in Fig. 1, it is assumed that LNB and GNB are converted to Galβ1-3GlcNAc6P and Galβ1-3GalNAc6P via the PTS, respectively, and LBCZ_0230 intracellularly hydrolyzes them (Fig. 5A). The N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25), encoded by the LBCZ_0229 gene, is presumably able to deacetylate GlcNAc6P and GalNAc6P produced from Galβ1-3GlcNAc6P and Galβ1-3GalNAc6P, respectively. LBCZ_0228 encodes a homologous protein of D-galactosamine 6-phosphate deaminase/isomerase AgaS from E. coli,⁴⁷⁾ and LBCZ_0228 is predicted to produce D-tagatose 6-phosphate from D-galactosamine 6-phosphate for the D-tagatose 6-phosphate pathway, involving tagatose-6-phosphate kinase (EC 2.7.1.144; LBCZ_0465) and tagatose-1,6-bisphosphate aldolase (EC 4.1.2.40; LBCZ_0466 and/or LBCZ_2366).²⁸⁾⁴⁸⁾ D-Glucosamine 6-phosphate is presumably converted to D-fructose 6-phosphate by glucosamine 6-phosphate isomerase (EC 3.5.99.6), encoded by LBCZ_2712, for the metabolism through the glycolysis. D-Galactose is predicted to be metabolized to α-D-glucose 1-phosphate through the Leloir pathway, involving galactokinase (EC 2.7.1.6; LBCZ_0451), UDP-glucose-hexose-1-phosphate uridylyltransferase (EC 2.7.7.12; LBCZ_0453), and UDP-glucose 4-epimerase (EC 5.1.3.2; LBCZ_0452), for further metabolism via the glycolysis. Although LBCZ_0230 possesses neither a typical signal sequence nor membrane anchoring motifs, this enzyme might extracellularly hydrolyze LNB/GNB or glycans that contain LNB/GNB structures as S. pneumoniae BgaC (Fig. 5B). BgaC also does not possess any sequence motifs indicating extracellular localization, but is indicated to be localized at the cell's surface.²⁰⁾²¹⁾ In addition to its high stability under acidic conditions, LBCZ_0230 exhibits high activity over a wide pH range (Fig. 3). These properties are suitable for the extracellular activity. GlcNAc and GalNAc, extracellularly produced from LNB and GNB by the β-galactosidase reaction, respectively, could be metabolized via the PTS pathway (Fig. 5B). L. casei JCM 1134 presumably utilizes D-galactose as in L. casei 64H, which utilizes D-galactose through a permease/Leloir pathway and a PTS/D-tagatose 6-phosphate pathway.⁴⁹⁾

Fig. 5. Possible functions of LBCZ_0230 in LNB/GNB metabolism.

　(A) Intracellular hydrolysis of phosphorylated LNB/GNB. Galβ1-3GlcNAc6P and Galβ1-3GalNAc6P are produced through uptake of LNB and GNB via the PTS, respectively, and LBCZ_0230 intracellularly hydrolyzes them. (B) Extracellular hydrolysis of LNB and GNB. LBCZ_0230 extracellularly hydrolyzes LNB and GNB, and produced GlcNAc and GalNAc are imported to the cytosol via the PTS.

LBCZ_0230's substrate binding mechanism to LNB has been predicted by superimposition of LBCZ_0230 onto A. niger β-galactosidase E298Q in complex with Galβ1-3Glc (Figs. 4C and D), and aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458 are expected to interact hydrophobically with the GlcNAc residue of LNB at subsite +1. Among these residues, Trp240, Trp243, and Tyr458 of LBCZ_0230 correspond to the essential residues for substrate binding in S. pneumoniae BgaC, Trp240, Trp243, and Tyr455,³⁸⁾ respectively. β-Galactosidases acting on β-galactosides harboring LNB/GNB structure contain aromatic residues at these positions (Fig. 6), and substitution of these residues with other aromatic residues yields greater activity for LNB than substitution with Ala.³⁸⁾ In addition to these aromatic residues, Trp190 and Phe244 also possibly have hydrophobic interactions with LNB. Notably, these residues are well conserved within the LNB/GNB hydrolyzing β-galactosidases (Fig. 6).

Fig. 6. Multiple sequence alignment of GH35 enzymes.

　The multiple alignment was constructed using MAFFT version 7,⁵⁰⁾ and visualized using ESPript 3.⁵¹⁾ The residues which are predicted to interact with LNB, are indicated by black circles. L_casei_JC, LBCZ_0230 (GenBank ID, BAN73398.1); L_casei_BL, GnbG (CAQ65417.1); S_pneumoni, S. pneumoniae β-galactosidase (AAK74249.1); S_suis, Streptococcus suis β-galactosidase (ABP89415.1); A_muciniph, A. muciniphila β-galactosidase (ACD04606.1); B_circulan, Bacillus circulans β-galactosidase (BAA21669.1); B_thetaiot, Bacteroides thetaiotaomicron β-galactosidase (AAO79265.1); C_japonicu, Cellvibrio japonicus β-galactosidase (ACE85180.1); P_thiamino, Paenibacillus thiaminolyticus β-galactosidase (CAZ44333.1); X_campestr, Xanthomonas campestris β-galactosidase (AAM42167.1); A_niger, Aspergillus niger β-galactosidase (AAE23229.1); A_oryzae, Aspergillus oryzae β-galactosidase (BAE60622.1); T_reesei, Trichoderma reesei β-galactosidase (CAD70669.1); S_lycopers, Solanum lycopersicum β-galactosidase (AAC25984.1); H_sapiens, Homo sapiens β-galactosidase (AAA51819.1). LBCZ_0230, GnbG, S. pneumoniae β-galactosidase, S. suis β-galactosidase, A. muciniphila β-galactosidase, B. circulans β-galactosidase, and H. sapiens β-galactosidase hydrolyze β-(1→3)-galactosidic linkage in LNB/GNB and glycan including the LNB/GNB structure. The sequences of A_niger, A_oryzae, and T_reesei are not shown in the lower alignment because their spatial location is apparently different from those of the others. Trp806 of A_niger locates at the corresponding position of Tyr458 of LBCZ_0230 as shown in Fig. 4C.

In this study, we demonstrate that GH35 LBCZ_0230, encoded by the gene in the operon of the PTS pathway of LNB/GNB metabolism, is a β-galactosidase with high activity towards LNB/GNB. We proposed the following two possible pathways for LNB/GNB metabolism involving LBCZ_0230: 1) LNB and GNB are imported to the cytosol via the PTS, and LBCZ_0230 intracellularly hydrolyzes Galβ1-3GlcNAc6P and Galβ1-3GalNAc6P; 2) LBCZ_0230 extracellularly hydrolyzes LNB, GNB, and glycan including LNB/GNB structure, and the PTS uptakes GlcNAc and GalNAc produced. For a better understanding of LNB/GNB metabolism via the PTS pathway, it is necessary to investigate subcellular localization of LBCZ_0230 and the activity of LBCZ_0230 to the phosphorylated LNB and GNB.

CONFLICTS OF INTEREST

The authors declare that they have no competing interests.

ACKNOWLEDGMENTS

We thank Dr. Mamoru Nishimoto of National Agriculture and Food Research Organization and Professor Motomitsu Kitaoka of Niigata University for providing LNB and GNB. We also thank Mr. Yusuke Takada of the DNA sequencing facility of the Research Faculty of Agriculture, Hokkaido University for assistance with DNA sequence analysis, and Ms. Nozomi Takeda of the Global Facility Center, Hokkaido University for the amino acid analysis. Strain JCM1134 was provided by Japan Collection of Microorganisms, RIKEN BRC which is participating in the National BioResource Project of MEXT, Japan. The synchrotron radiation experiments were performed at the BL44XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2018A6810, 2017B6713, 2017A6713, 2016B6611 and 2016A6611). We thank the staff of the beamline BL44XU at Spring-8 for their assistance during data collection. We would like to thank Editage (www.editage.com) for English language editing.

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