# Novel $\beta$-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154

Lisanne Hameleers, Tjaard Pijning, Brandon B Gray, Régis Fauré, Edita Jurak

► To cite this version:

Lisanne Hameleers, Tjaard Pijning, Brandon B Gray, Régis Fauré, Edita Jurak. Novel $\beta$-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154. New Biotechnology, 2024, 80, pp.1-11. 10.1016/j.nbt.2023.12.011. hal-04670505

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# Novel β-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154

Lisanne Hameleers<sup>a</sup>, Tjaard Pijning<sup>b</sup>, Brandon B. Gray<sup>a</sup>, Régis Fauré<sup>c</sup>, Edita Jurak<sup>a, *</sup>

## ARTICLE INFO

Keywords:

β-Galactosidase
Glycoside Hydrolase family 154
(α/α)<sub>6</sub> Topology
Carbohydrate Active enZymes
Polysaccharide Utilization Loci
Enzyme discovery

## ABSTRACT

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo-β-galactosidase activity with regiopreference for hydrolyzing β-D-(1,6) glycosidic linkages. Notably, it exhibited a preference for D-glucopyranosyl (D-Glcp) over D-galactopyranosyl (D-Galp) and D-fructofuranosyl (D-Fruf) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-β-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (α/α)<sub>6</sub> topology, which has not been observed before for β-galactosidases. BD-β-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-β-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from *Bacteroides thetaiotaomicron*, described as β-glucuronidase, displayed exo-β-galactosidase activity like BD-β-Gal but lacked a tetrameric structure.

## 1. Introduction

In the era of low-cost sequencing methods, nucleotide sequence databases have become rich sources for enzyme discovery because functional annotation of around 40–50 % of sequenced genomes is still absent [1–4]. Carbohydrate Active enZymes (CAZymes) are interesting targets with diverse applications, including biomass valorization and nutritional products such as galactooligosaccharides (GOS) and lactose free milk [5–7]. They are classified in the CAZY database (www.cazy.org/ [8]) and a successful strategy for their discovery is the investigation of Polysaccharide Utilization Loci (PULs). These are physically linked gene clusters frequently found in the genomes of the Bacteroidota phylum (previously named Bacteroidetes [9]) [10–12]. They encode an array of CAZymes, Carbohydrate Binding Modules (CBMs), Surface

Glycan Binding Proteins (SGBP), carbohydrate transporters and Proteins of Unknown Function (PUFs) that are simultaneously upregulated in the presence of a specific carbohydrate. The value of PUL-driven enzyme discovery has been proven by abundant studies characterizing the synergistic degradation of numerous substrates [13–23]. PUL-encoded PUFs are high-risk but high-gain targets that could open new starting points for functional prediction. The selected candidate, now named BD-β-Gal, was a PUF before it was classified as Glycoside Hydrolase family 154 (GH154). The GH154 family was, with 4029 sequences (November 2023), large but only the founding member BT3677 has been functionally characterized as exo-β-(1,6)-D-glucuronidase and no crystal structure was reported [22]. Due to low sequence identity between BT3677 and BD-β-Gal (25.1 %) we proceeded with the characterization of BD-β-Gal to improve the functional prediction of the members of

Abbreviations: PUL, Polysaccharide Utilization Locus; CAZyme, Carbohydrate Active enZyme; PUF, Protein of Unknown Function; GH, Glycoside Hydrolase; D-Glcp, D-glucopyranosyl; D-Galp, D-galactopyranosyl; D-Fruf, D-fructofuranosyl; pNP, para-nitrophenyl.

<footnote><sup>a</sup> Department of Bioproduct Engineering, Engineering and Technology institute Groningen (ENTEG), University of Groningen, Nijenborgh 4, Groningen 9747 AG, the Netherlands</footnote>

<footnote><sup>b</sup> Department of Biomolecular X-ray Crystallography, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, Groningen 9747 AG, the Netherlands</footnote>

<footnote><sup>c</sup> TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: e.jurak@rug.nl (E. Jurak).</footnote>
GH154.

In this work, we characterized BD-β-Gal as a β-galactosidase (E.C. 3.2.1.23), a new catalytic activity for the GH154 family, and determined its crystal structure with a topology not previously observed for β-galactosidases. Further, we describe novel β-galactosidase activity of BT3677, the founding member of GH154.

## 2. Materials and methods

### 2.1. Substrates and chemicals

All para-nitrophenyl (pNP)-glycosides and substrates of the initial screening (Suppl. Table S1), all galactobiose, β-D-galactopyranosyl-(1,3)-D-glucose, and β-D-galactopyranosyl-(1,6)-D-glucose were from Megazyme Ltd. (Bray, Ireland). All chemicals were from Sigma-Aldrich (St. Louis, MO, USA). Gene synthesis was performed by GenScript (Piscataway, NJ, USA). *E.coli* BL21 (DE3) strains were obtained from New England BioLabs Inc. (Ipswich, MA, USA), MagicMedia™ and Pierce BCA Protein Assay from Thermo Fisher Scientific (Sunnyvale, CA, USA). SDS-PAGE reagents were from BioRad (Hercules, CA, USA).

### 2.2. Heterologous protein expression and purification

BD-β-Gal (45181_263) which was identified in a published meta-
genome from beaver droppings [24] was selected for characterization
and BT3677 (UniProt: Q8A114_BACTN) from B. thetaiaotomicron was
included for comparison [22]. Signal peptide and domain prediction was
performed with SignalP6.0 [25] and InterPro [26,27]. E. coli codon
optimized sequences, excluding the predicted signal sequence, were
synthesized between the Xhol and Ndel restriction sites of a pET29b(+)
plasmid containing a C-terminal His-tag. The BD-β-Gal E73A mutant
expression vector was synthesized similarly. Following transformation
of E.coli BL21 (DE3), 500 mL MagicMedia™ cultures were incubated
following the manufacturer's protocol for protein production. The cells
were harvested at 5000 x g for 10 min at 4 °C, resuspended in 50 mL of
20 mM HEPES (BD-β-Gal) or 20 mM sodium phosphate (BT3677) both at
pH 7.4 and containing 500 mM NaCl, 20 mM imidazole, and frozen at
-20 °C. After thawing, cells were sonicated on ice twice for 5 min at 50 %
duty cycle (Sonics & Materials-Vibra Cell, Newtown, CT, USA) and the
supernatant was clarified by centrifugation at 12,000 x g for 15 min at
4 °C. The recombinant proteins were purified by immobilized metal
affinity chromatography using a 1 mL His-Trap™ HP column (Cytiva,
Marlborough, MA, USA) on an ÄKTA system (GE Healthcare, Chicago,
IL, USA). The column manufacturer's protocol was followed using 250
mM imidazole for elution. The presence of the desired protein in the
fractions was confirmed by SDS-PAGE analysis (see supplementary
material). Pooled fractions were buffer exchanged into 20 mM HEPES,
pH 7.4 (BD-β-Gal and BD-β-Gal E73A) or 20 mM sodium phosphate
buffer, pH 7.4 (BT3677) using VivaSpin® 20 ultrafiltration units (30,
000 MWCO, Satorius AG, New York, NJ, USA). Drops of buffer
exchanged enzyme were flash frozen in liquid nitrogen for storage at
-80 °C. Protein concentration was determined with Pierce BCA Protein
Assay. Protein purity was assessed by SDS-PAGE and Dynamic Light
Scattering (DLS) on a DynaPro NanoStar (Wyatt, Santa Barbara, USA) at
293 K; the samples contained 10 mg/mL BD-β-Gal in 20 mM HEPES, pH
7.4, 150 mM NaCl or 5.8 mg/mL BT3677 in 20 mM sodium phosphate,
pH 7.4, 100 mM NaCl.

### 2.3. Initial screening

Two initial screenings to detect Glycoside Hydrolase (GH) activity of BD-β-Gal were conducted according to previous work [15]. In 96-well plates, 160 µL of a substrate (SI, SII, specified below) and 40 µL enzyme (EI, EII; specified below) were mixed and incubated at 40 °C under 150 rpm, sealed with plastic foil to prevent evaporation (S+E). These incubations were performed in three 50 mM buffers, specifically

sodium acetate, pH 5.5; HEPES, pH 7.0; and HEPES, pH 8.5. For each condition, three independent incubations were prepared and stopped after 2, 4, and 24 h (n = 3). As substrate blanks (SBL), 40 µL buffer was added instead of enzyme (n = 2). As standards, dilutions of pNP (0–0.3 mM), D-xylose, or D-glucose (both 0–0.1 mg/mL) in milli-Q water were used. Product release was measured at an absorbance of 405 nm using a spectrophotometer (SpectraMax Plus, Molecular Device, San Jose, CA, USA). For the pNP-screening, ten pNP-glycosides (Suppl. Table S1) were dissolved in DMSO to 50 mM and diluted to 1.25 mM in all three tested reaction buffers (SI). Freshly thawed enzyme was diluted to 2.5 mg/mL in all three buffers (EI). After mixing SI and EI, a final concentration of 1 mM substrate and 0.5 mg/mL (100 µg) enzyme was obtained. To stop the reactions and ensure alkaline conditions in each well 50 µL of 725 mM Na₂CO₃ were added before measuring pNP release. For the PAHBAH reducing-end screening [28,29], three hydrolyzed polysaccharides (5 mg/mL in 20 mM sodium citrate, pH 6) and fourteen polysaccharides (10 mg/mL in water; Suppl. Table S1) were diluted to 0.75 mg/mL in all three buffers (SII). Freshly thawed enzyme was diluted to 0.3 mg/mL in all three buffer (EII). After mixing SII and EII, a final concentration of 0.6 mg/mL substrate and 0.06 mg/mL (12 µg) enzyme was obtained. To stop the reaction the 96-well plates were boiled for 10 min at 100 °C in a water bath. A change in reducing ends was determined by mixing 50 µL of each well with 200 µL freshly prepared PAHBAH-reagent, composed of a 1:4 ratio of 0.5 M NaOH: 5% w/w p-hydroxybenzhydrazide in 0.5 M HCl, in a 96-well plate that was incubated at 70 °C for 30 min before measuring absorbance at 405 nm. For both assays, the average absorbance of SBL was subtracted from the corresponding absorbance of S+E. Appropriate standard curves were used to quantify product release.

### 2.4. Determination of pH- and temperature optima

The pH and temperature optima were determined under the same conditions as described for the initial pNP-screening with the following adjustments. As substrate pNP-β-D-Galp was used, Na₂CO₃ was added after 1 h, and all incubations were centrifuged at 20,000 x g for 2 min before measuring product release to ensure removal of protein precipitation observed at low pH and high temperature. For the pH optima 50 mM buffers, specifically sodium citrate at pH 4.0–5.0, sodium phosphate at pH 6.0–8.5, and glycine sodium hydroxide at pH 8.5–11.0 were used. The temperature optimum was determined between 10 and 80 °C in 50 mM sodium phosphate buffer at pH 7.0.

### 2.5. Determination of kinetic parameters and specific activity

The kinetic parameters were determined using the assay described for the initial pNP-screening with the following adjustments. Reactions were performed in triplicate at 50 °C in 10 mM sodium phosphate buffer pH 7.0 using six pNP-β-D-Galp concentrations (0.05–5.0 mM). In independent plates for six time points between 5–60 min, product release was determined. The initial reaction rates (v₀) [nmol/min] were plotted as a function of substrate concentrations [mM] and fitted using the Michaelis-Menten equation in Origin 8.1 (Northampton, MA, USA) to calculate K_m [mM], v_max [nmol/min] and k_cat [s⁻¹]. One unit [U] of β-galactosidase activity was defined as the amount of enzyme needed to release 1 µmol pNP per min from pNP-β-D-Galp at 50 °C in 10 mM sodium phosphate buffer pH 7.0. Specific activities [U/mg] were calculated by dividing v_max by initial enzyme concentration (0.1 mg).

### 2.6. Enzyme activity on dimeric substrates

In reaction tubes, 0.2 mU BD-β-Gal (100 µg) and 0.2 mU BT3677 (377 µg) were incubated on 0.3 mM β-(1,4)-galactobiose, β-(1,6)-galactobiose, β-D-Galp-(1,3)-D-Glcp, β-D-Galp-(1,4)-D-Glcp (lactose), β-D-Galp-(1,6)-D-Glcp (allolactose), and β-D-Galp-(1,4)-D-Fruf (lactulose) in 10 mM sodium phosphate buffer pH 7.0 at 40 °C under 150 rpm shaking in 200 µL for 24 h. BD-β-Gal was additionally incubated on β-(1,3)-
galactobiose. SBLs were used as controls. All samples were centrifuged at 20,000 x g for 2 min before analyzing with High Performance Anion Exchange Chromatography (HPAEC) coupled to a Pulsed Amperometric Detector (PAD) on a Dionex Ultimate 6000 system with Chromeleon 7.3.1 software (Thermo Fisher Scientific). Mono- and oligosaccharide separation was achieved on a Dionex CarboPac PA-1 IC and PA-300 IC column (guard- and analytical, all 2 mm x 250 mm). As eluents 100 mM NaOH (A), 1 M NaOAc in 100 mM NaOH (B), 200 mM NaOH (C), and milliQ water (D) were used. On PA-1 column elution was achieved with an initial flow of 100 % A at 0.3 mL/min, followed by 35 min linear gradient up to 38 % B, and 8 min plateau of 100 % B. On PA-300 column elution was achieved with an initial flow of 100 % C at 0.25 mL/min, followed by 8 min at 5 % C/ 95 % D, 7 min linear gradient up to 100 % C, and 9 min at 100 % C. Results on the PA-1 column were used for Table 1; PA-300 column results are in Fig. S6.

## 2.7. Crystallization and data collection

Initially, a sitting drop crystallization screening was performed in 96-well plates using a Mosquito crystallization robot (SPT Labtech, Melbourn, UK) and commercially available screens, giving several hits. Subsequent steps involved optimization of a few of these hits in sitting drops and, in the final stages, hanging drops, which were setup manually in 12-well plates. In the final optimized condition, based on condition 31 (C7) from the SG-1 screen (Molecular Dimensions, Rotherham, UK), the protein solution contained 10 mg/mL protein, 20 mM HEPES, pH 7.4, 150 mM NaCl; reservoir solutions contained 16 % (w/v) PEG 3350, 100 mM HEPES, pH 7.5, 150 mM NaCl. Hanging drops were prepared by mixing 1.5 µL protein solution and 1.5 µL reservoir solution, and equilibrated at 293 K. Prior to data collection, crystals were briefly transferred to 25 % (w/v) PEG 3350, 100 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 15 % (v/v) glycerol (GOL), and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at beamline ID23-1 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and processed using EDNA [30]. Statistics are given in Suppl. Table S2.

## 2.8. Structure determination and refinement

The BD-β-Gal crystal structure was determined by molecular replacement using PHASER [31] and an AlphaFold2-generated template [32,33]. Refinement and model building was carried out using Refmac [34], COOT [35] and (in the final stages) PDB-REDO [36]. Suppl. Table S2 lists the refinement statistics and model quality. Structural figures were prepared with PyMOL [37]. DSSP was used to analyze the secondary structure [38]. Analysis of structural similarity was

<caption>**Table 1**</caption>

Comparison of relative activities of BD-β-Gal and BT3677 on disaccharides containing D-galactopyranosyl (D-Galp) at their non-reducing end.

<table><thead><tr><th>Substrate</th><th>BD-β-Gal<sup>a</sup></th><th>BT3677<sup>a</sup></th></tr></thead><tbody><tr><td></td><td>Relative activity<br/>[%]<sup>b</sup></td><td>Relative activity<br/>[%]<sup>b</sup></td></tr><tr><td>β-(1,3)-galactobiose</td><td>active<sup>c</sup></td><td>n.a.</td></tr><tr><td>β-(1,4)-galactobiose</td><td>n.d.</td><td>n.d.</td></tr><tr><td>β-(1,6)-galactobiose</td><td>85 ± 0.6</td><td>66 ± 3.0</td></tr><tr><td>β-D-Galp-(1,3)-D-Glc<sup>p</sup></td><td>70 ± 0.7</td><td>55 ± 2.0</td></tr><tr><td>β-D-Galp-(1,4)-D-Glc<sup>p</sup> (lactose)</td><td>84 ± 0.4</td><td>59 ± 6.2</td></tr><tr><td>β-D-Galp-(1,6)-D-Glc<sup>p</sup><br/>(allolactose)</td><td>100 ± 3.2</td><td>100 ± 5.2</td></tr><tr><td>β-D-Galp-(1,4)-D-Fruf (lactulose)</td><td>19 ± 1.0</td><td>n.d.</td></tr></tbody></table>

n.d. not detected, n.a. not analyzed

<sup>a</sup> 0.2 mU of enzyme was incubated on 0.3 mM substrate in 10 mM phosphate buffer pH 7.0 at 40 °C for 24 h in 200 µL; n = 3.

<sup>b</sup> Peak areas of D-Galp, D-Glc<sup>p</sup>, D-Fruf were quantified on HPAEC-PAD using PA-1 column using monosaccharide standard curves. Results were normalized to highest observed values = activity on allolactose.

<sup>c</sup> n = 1 due to limited substrate availability

performed (October 2022) using PDBeFold [36] and Foldseek [39]. The PDBePISA webserver [40] was used to analyze molecular interfaces in the crystal structure. The ConSurf webserver [41] was used with default parameters to analyze the distribution of residue conservation and to identify the putative active site. Atomic coordinates and structure factors of BD-β-Gal have been deposited at the Protein Data Bank with entry 8O14.

## 2.9. AlphaFold modeling of BT3677

AlphaFold2 [32,33] was used to generate a model for BT3677 from *B. thetaiotaomicron* (UniProt: Q8A114_BACTN). Residues with a per-residue confidence score (pLDDT) >60 (residues 31–673) were used in structural comparisons with BD-β-Gal. The AlphaFold model for BT3677 showed predicted Local Distance Difference Test values above 65 for almost all (32–673) of its 673 residues, indicating that except for the N-terminal residues (including the signal peptide), reliable modeling was obtained.

## 2.10. Studying conserved residues

Redundancy of all 2444 sequences (November 2021) in GH154 family was reduced using CD-HIT [42] with 95 % threshold resulting in a clustered dataset with 996 sequences. To identify conserved residues within these 996 sequences a Multiple Alignment using Fast Fourier Transform (MAFFT) with local alignment settings was used [43]. Conserved residues were visualized with a sequence logo created with WebLogo [44].

# 3. Results and discussion

## 3.1. Initial screenings and optimal conditions

Information about the genetic location, sequence homologies and cellular localization of BD-β-Gal and BT3677 were gathered by in silico studies. BD-β-Gal was encoded in BD-PH PUL127 of a previously studied metagenome of Beaver Droppings (BD) [24] and BT3677 was encoded in the short PUL<sub>AGPS</sub> of *B. thetaiotaomicron* [22]. Their PUL architecture was not conserved but both encoded proteins with identical annotation (Fig. 1). Further, both were expected to be secreted as they contain a predicted signal sequence. Remarkably, their N-terminal DUF2264 (PF10022) domains share only 25 % sequence identity with a 74 % query cover and an e-value: 1e-25, despite their common membership within the GH154 family. This highlights the considerable sequence diversity that exists among members of the same CAZY family. Next to the DUF2264, BT3677 has an additional C-terminal domain (residues 410–673) with no domain affiliation (Fig. 2A, Suppl. Fig. S1). The fact that BT3677, the only homolog of BD-β-Gal with described catalytic activity, showed such sequential differences motivated the functional and structural characterization.

The purity and actual molecular weights of recombinant BD-β-Gal and BT3677 were confirmed by SDS-PAGE and DLS (Fig. 2B, Suppl. Fig. S2). When characterizing a PUF, detecting any catalytic activity is the first invaluable step. Therefore, an initial screening at 3 pH values and on a broad substrate library was performed. The observed pNP release from pNP-β-D-Galp was promising and suggested β-galactosidase activity for BD-β-Gal (Suppl. Fig. S3). Interestingly, BT3677 was capable of hydrolyzing pNP-β-D-Galp but no hydrolytic activity on either pNP-β-D-Glc<sup>p</sup>A or pNP-α-D-Glc<sup>p</sup>A was detected when measured (data not shown). We propose that the minimal structure of the synthetic substrate pNP-β-Glc<sup>p</sup>A in comparison to the naturally derived tetrasaccharide used in [22] and/or the different stereochemistry of the glycosidic bond of pNP-α-Glc<sup>p</sup>A may account for the lack of detectable activity on these artificial substrates.

With the identification of pNP-β-D-Galp as potential substrate, the pH and temperature optima of BD-β-Gal and BT3677 were determined and
BD-PH PUL127

Metagenome from Beaver droppings

<figure><img src="image_3.png" /></figure>

PUL<sub>AGPS</sub> (BT3674-BT3687)

*Bacteroides thetaotaomicron*

<figure><img src="image_4.png" /></figure>

**Fig. 1.** Schematic of Polysaccharide Utilization Loci (PULs) encoding BD-β-Gal and BT3677. Annotations: SusC-like: Ton-B dependent outer membrane transporter (purple), SusD-like: outer membrane binding protein (orange), GH: Glycoside Hydrolase (light pink), PL: Polysaccharide Lyase (pink), CBM: Carbohydrate Binding Module (green), and PUF: Protein of Unknown Function (grey, named unknown (UNK) in PULDB, www.cazy.org/ PULDB/ [45]), red bars indicate the margins of assembled region.

<figure><img src="image_5.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_7.png" /></figure>

**Fig. 2.** Domain architecture (A) and 12 % SDS-PAGE (B) of 46 kDa BD-β-Gal and 75 kDa BT3677. Molecular weight marker [kDa] (1), 10 µg (2) and 1 µg (3) BT3677, 10 µg (5) and 1 µg (6) BD-β-Gal.

found to be pH 7.0 and 50 °C for both enzymes (Fig. 3). The temperature and pH optima of other β-galactosidases range from 10 to 70 °C and from pH 4.0 to 8.5, as they are highly dependent on the origin of the host organism [46–50]. The relative activities of both enzymes decreased to below 50 % at pH ≤5.0 and ≥9.5, and at temperatures ≤30 and ≥60 °C (Fig. 3). Since it is known that divalent cations can affect the hydrolytic activity of β-galactosidases [51,52], the effect of nine divalent cations and EDTA was tested. As has been previously reported [51–53], the addition of MgSO₄ (10 mM) increase the β-galactosidase activity on pNP-β-D-Galp for both BD-β-Gal and BT3677, while all other cations and EDTA inhibited their activity (Fig. S4). Based on the obtained results, we conclude that BD-β-Gal and BT3677 are capable of hydrolyzing pNP-β-D-Galp at the given conditions, demonstrating exo-β-galactosidase activity.

## 3.2. Biochemical characterization and kinetic parameter

At optimal conditions, BD-β-Gal and BT3677 showed turnover numbers ($k_{cat}$) of 1.04 and 2.50 s⁻¹ (Suppl. Fig. S5). These values were in the same order of magnitude if compared to other β-galactosidase, including YesZ from *Bacillus subtilis* (1.58 s⁻¹ [54]) and the commercially used β-galactosidase from *Kluyveromyces lactis* (3.49 s⁻¹ on oNP-β-D-Galp [55,56]). The maximum velocities ($v_{max}$) of BD-β-Gal and BT3677 were, with 0.13 ± 0.01 and 0.20 ± 0.00 nmol/min, respectively, very similar (Fig. S5). The $K_m$ values of BD-β-Gal (0.11 ± 0.03 mM) and BT3677 (0.28 ± 0.02 mM) were found to be in the same order of magnitude as that of the recombinant RsBGAL1 from radish (*Raphanus sativus*, 0.33 mM [46]). Thus, both enzymes displayed Michaelis-Menten behavior (Suppl. Fig. S5). Specific activities of BD-β-Gal and BT3677 on pNP-β-D-Galp at 50 °C were determined to be 1.3 and 2.0 mU/mg, respectively.

To investigate substrate acceptance of BD-β-Gal and BT3677 beyond

pNP-β-D-Galp, disaccharides consisting of D-Galp, D-Glcp, and/or D-Fruf were used for assays (Fig. 4A). The results demonstrated that both enzymes hydrolyzed β-(1,3)- and β-(1,6)-galactobiose, β-D-Galp-(1,3)-D-Glcp, lactose, and allolactose, while β-(1,4)-galactobiose remained intact (Fig. 4B, Suppl. Fig. S6). The seemingly contradictory ability to act on lactose but not on β-(1,4)-galactobiose, despite their shared glycosidic linkage configuration, can be attributed to differences in the hydroxyl group configuration of the C-4 epimers D-Galp and D-Glcp located at the reducing end of these two substrates (Fig. 4A). The indicated hydroxyl group at the C-4 position is involved in the glycosidic bond of the disaccharides, leading to different spatial orientations of the hexopyranose rings and thus differences in substrate binding. Such substrate specificity, has been reported for other β-galactosidases [46,57].

The highest relative activity for both enzymes was observed when incubated on allolactose, with BD-β-Gal completely hydrolyzing the substrate (Table 1, Fig. 4B). Only BD-β-Gal hydrolyzed lactulose, albeit with a low relative activity of 19 % suggesting a low preference for this substrate (Table 1). Transglycosylation activity on lactose was observed for both enzymes detected by the formation of allolactose (Fig. 4B). We expected that the produced allolactose was readily hydrolyzed, explaining the small detected peak size (Fig. 4B). BD-β-Gal hydrolyzed pNP-β-D-Galp but not pNP-β-D-Glcp; thus it can be inferred that BD-β-Gal and BT3677 cleave substrates with a D-Galp unit at the non-reducing end, and that D-Glcp is preferred over D-Galp at the reducing-end of disaccharides. Consequently, both BD-β-Gal and BT3677 are in fact exo-β-galactosidases. Moreover, BD-β-Gal and BT3677 showed regiopreference for the hydrolysis of β-D-(1,6) linkages, along with the ability to hydrolyze other glycosidic linkages, including the one present in β-D-(1,3)-galactobiose (Table 1).

The similar genomic location of BD-β-Gal and BT3677 within a PUL, suggested that the observed β-galactosidase activity of BD-β-Gal might support the degradation of the branched β-D-Galp-(1,3)-[β-D-Galp-(1,6)]-β-D-Galp structure of arabinogalactan (Acacia tree gum) together with the enzymes encoded in the same PUL, as it has been described for BT3677 [22]. Based on this previous research, it could be speculated that a dual β-galactosidase and β-glucuronidase could have a synergistic effect during the degradation of AG. As far as we are aware, no natural bifunctional β-galactosidase/β-glucuronidase has been reported yet but a β-galactosidase from *Pyrococcus woesei* was successfully converted into a β-glucuronidase by directed evolution suggesting that these two catalytic activities are closely related [58,59].

## 3.3. Overall crystal structure of BD-β-Gal and AlphaFold model of BT3677

The BD-β-Gal crystal structure was determined at a resolution of 1.76 Å in space group *P* 2₁, with the asymmetric unit containing four protomers (chains A-D) forming a homotetramer (Fig. 5A). BD-β-Gal displays a barrel-like ($α/α_6$) topology with six inner helices surrounded
<figure><img src="image_8.png" /><figcaption>Fig. 3. pH and temperature profiles of BD-β-Gal (A, C) and BT3677 (B, D). 100 µg enzyme was incubated on 1 mM pNP-β-D-Galp in 50 mM buffer for 1 h. After centrifugation and addition of Na₂CO₃, absorbance was measured at 405 nm. pH profiles were determined at 40 °C; temperature profiles at pH 7. Red squares: citrate buffer pH 4–6, blue circles: sodium phosphate buffer pH 7–8, orange triangles: glycine sodium hydroxide buffer pH 8–11 (n = 3).</figcaption></figure>

by six outer α-helices (Fig. 5B). Remarkably, one of the inner helices is a 3₁₀-helix and another one is heavily distorted featuring 3₁₀-helical elements (Fig. 5B). Notably, between the C-terminal ends of the helices forming the inner helix bundle, a central cavity is found, facing outward from the tetramer (Fig. 5A,C). PDBeFold and Foldseek analysis and structural comparison revealed that the (α/α)₆ topology of BD-β-Gal shows similarity to certain GHs and Polysaccharide Lyases (PLs), but with low overall sequence identity (Suppl. Table S3) and with clear differences on a more detailed level. Interestingly, structurally characterized β-galactosidases (GH1, GH2, GH35, GH42, GH59) instead display a (β/α)₈ topology. Thus, the (α/α)₆ topology of BD-β-Gal, a member of GH154, represents a novel fold for β-galactosidases. The (α/α)₆ topology is conserved in the BT3677 AlphaFold model; the two can be superimposed with RMS (root mean square deviation) of 1.87 Å for 231 Cα atoms. Notably, however, BT3677 has an additional C-terminal domain (residues 410–673), with a supersandwich topology. Since the extra C-terminal domain packs against the same side of the (α/α)₆ domain that provides dimer contacts in BD-β-Gal, BT3677 cannot form a similar tetrameric arrangement (Suppl. Fig. S1). DLS analysis indeed showed that BT3677 is monomeric, with an estimated molecular mass of 60 kDa (in regularization analysis), somewhat lower than the

theoretical value of a monomer (75 kDa; Suppl. Fig. S2).

### 3.4. The putative active site

Even though the BD-β-Gal crystal structure has no carbohydrate ligands bound, it is tempting to assign its central cavity as its active site (Fig. 5B,C); however since the GH154 BD-β-Gal displays a different topology and only shares low sequence homology with canonical β-galactosidases this is not trivial, and underpins the novelty of the GH154 BD-β-Gal structure. Nevertheless, PDBeFold analysis listed GHs and PLs as closest hits (Suppl. Table S3) of which some feature a carbohydrate ligand bound in their central cavity that lies between the C-terminal ends of the inner α-helices and helical barrel. Furthermore, each BD-β-Gal protomer in the crystal structure has one or two glycerol molecules (GOL) in the central cavity. Glycerol resembles part of a pyranose ring structure and is often observed in carbohydrate binding sites. Finally, ConSurf analysis revealed that within the GH154 family residues surrounding the central cavity constitute the most conserved region (Fig. 6A) including the fully conserved residues E73, R77, Q129 and Y296 and the almost conserved D133, which is sometimes replaced by a glutamate (Fig. 6B,C). All residues contribute to an extensive hydrogen-
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** β-galactosidase and transglycosylation activity of BD-β-Gal and BT3677 on D-Galp-containing disaccharides. (A) Symbol nomenclature and chemical structures. (B) 0.2 mU of BD-β-Gal or BT3677 were incubated on the indicated substrates (0.3 mM) in 10 mM sodium phosphate buffer pH 7.0 at 40 °C for 24 h. Product formation was analyzed on HPAEC-PAD with PA-300 column (representative chromatogram of *n* = 3).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Crystal structure of BD-β-Gal. (A) BD-β-Gal homotetramer showing side-by-side arrangement with the central cavities alternatively pointing up (chains A and C) or down (chains B and D). (B) Cartoon representation of one protomer (chain A) highlighting the (α/α)₆ topology, with an inner helix bundle (α- and 3₁₀-helices shown in purple or pink, resp.) surrounded by outer helices (α- and 3₁₀-helices shown in red or orange, resp.). The N- and C-terminal residues are indicated with a blue resp. red sphere; the two glycerol (GOL) molecules bound in the central cavity are shown in stick representation with yellow carbon atoms. (C) Surface representation of chain A with GOL in central cavity.</figcaption></figure>

bonding network with the deepest bound glycerol molecule (GOL1) and water molecules (Fig. 7A).

β-Galactosidases from other GH families typically employ two acidic amino acid residues (D or E) to catalyze their reaction, initially suggesting that E73 and D133 could fulfil this role in BD-β-Gal. Their mutual distance of 4.4 Å would fit with a retaining mechanism observed in most canonical β-galactosidases [60]. Indeed a 58 % reduction in relative activity was observed for BD-β-Gal E73A mutant (Suppl. Fig. S7). However, while the E73 side chain protrudes into the cavity, the D133 side chain is buried behind R77 and Q129, which makes it unlikely to function as a catalytic residue. Notably, there is no other aspartate or glutamate within 12 Å of E73. Attempts to get more insights by obtaining *in crystallo* BD-β-Gal complexes were so far unsuccessful (possibly due to the presence of glycerol in the cryoprotectant). Preliminary docking experiments with the disaccharide β-(1,6)-galactobiose, however, did confirm that the bottom of the cavity does fit such substrates (data not shown). We also investigated whether BD-β-Gal could apply the novel catalytic mechanism described for the β-glucosidase 2 (GBA2; PDB: 5BVU), a member of GH116 with (α/α)₆ topology, but superposition of both structures was poor [61]. Taken together, our observations support the assignment of the central cavity as the active site of BD-β-Gal (Fig. 5B,C, Fig. 6A), but the BD-β-Gal mechanism remains unknown.

Compared to BD-β-Gal, the BT3677 AlphaFold model also features a central cavity, but with some differences: (a) semi-conserved replacement of D133<sup>BD-β-Gal</sup> by E158<sup>BT3677</sup>, (b) non-conserved replacements at the bottom and the rim of the cavity, and (c) a different loop arrangement of the entrance to the central cavity (Fig. 6C). Details of these differences are described in the supplementary files (Suppl. Fig. S8). It remains to be determined whether and how these differences contribute to the different reaction specificities of the two enzymes.

### 3.5. The BD-β-Gal tetramer

BD-β-Gal crystallized with four molecules in the asymmetric unit with close contacts provided by outer helices and loops (Fig. 5A). PDBePISA results indicated a stable tetramer suggesting that BD-β-Gal is

also tetrameric in solution (Suppl. Table S4), which is in agreement with the DLS experiments showing an estimated molecular mass of 165 kDa (in regularization analysis; Suppl. Fig. S2). The tetramer can be regarded as a dimer of dimers, with 29 % of the total surface area buried and the central cavities facing outwards in alternating up/down manner (Fig. 5A, Suppl. Table S4). Since none of the homologs found by PDBeFold and Foldseek had tetrameric structures (Suppl. Table S3), the BD-β-Gal tetramer seems to be unique.

For three of the four BD-β-Gal protomers, the α9-α10 loop of a neighboring protomer protrudes 'inwards' towards the central cavity, with residue R252 at the tip of this loop interacting with a bound GOL2 at the rim of the cavity (chains A and D; Fig. 7B). In contrast, for the fourth protomer, the neighboring α9-α10 loop is shifted upwards about 14 Å, out of the cavity, with no specific contacts to other protomers and lacking electron density for residues R252-D253. No glycerol is observed in the cavity either in this case. Together, these observations suggest that the α9-α10 loop is flexible in solution, and that the inward conformation plays a role during catalysis by binding carbohydrate substrates; thus, the tetrameric structure may be functional and confer unique specificity to BD-β-Gal compared to BT3677. In this light, R252 and R253 are interesting additional mutation targets outside the active site cavity.

### 4. Conclusion and outlook

With the presented β-galactosidase activity of BD-β-Gal, a new catalytic activity was introduced to the GH154 family and the first crystal structure was determined. BD-β-Gal and the founding member of GH154, BT3677, showed *exo*-β-galactosidase activity on *pNP-β-D-Galp*, β-(1,3)-galactobiose, β-(1,6)-galactobiose, *β-D-Galp*-*(1,3)-*D-Glc*p, lactose, and allolactose with a preference for β-(1,6)-linkages and *D-Glc*p over *D-Galp* as reducing-end sugar (glycon leaving group). BD-β-Gal also displayed low β-galactosidase activity on lactulose and both enzymes showed transglycosylation activity on lactose. The BD-β-Gal structure, with so far unobserved (α/α)₆ topology, may represent a novel β-galactosidase active site performing glycosidic bond cleavage, different from canonical β-galactosidases. We are currently targeting residues in BD-β-Gal (e.g. E73, R252, D253) for mutational studies in
<figure><img src="image_12.png" /><figcaption>**Fig. 6.** Putative active site of BD-β-Gal and its conserved residues. (A) ConSurf analysis of 996 GH154 sequences showing residue conservation (blue = high, red = low) mapped onto BD-β-Gal chain A. Viewed 'from the top' looking into the central cavity, with two bound glycerol molecules (GOL1–2) shown in stick representation and yellow carbon atoms. (B) Sequence logo of conserved residues of 996 GH154 sequences around the putative active site. (C) Residues surrounding the central cavity in BD-β-Gal and BT3677. Residues that were (almost) fully conserved among 996 GH154 sequences are underlined. Residues providing hydrogen bond interactions to GOL1 are indicated with an asterisk (*).</figcaption></figure>

order to gain more insight in this matter.

## Author's contribution

LH was involved in candidate selection, performed recombinant protein production, biochemical characterization, and major data analysis and was the leading author in writing the manuscript. TP performed all experiments for structural characterization of BD-β-Gal and obtaining the BT3677 AlphaFold model, he lead structural data analysis and wrote corresponding paragraphs. RF was involved in in-depth intellectual discussions, result interpretation and manuscript writing. BBG determined pH and temperature optima for BT3677 and helped with recombinant protein purification. EJ was involved in conceptualization,

candidate selection, data analysis, validation, and critical reviewing the manuscript. All authors have approved the final article.

## Funding

The University of Groningen financially supported this work (RuG Investment agenda/funds CvB Agrifood).

## CRediT authorship contribution statement

**Lisanne Hameleers:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing.
<figure><img src="image_13.png" /><figcaption>**Fig. 7.** Stereo views of the central cavity of the BD-β-Gal crystal structure with surrounding residues in stick representation. (A) Fully conserved residues are shown with orange carbon atoms, and the deepest bound glycerol molecule (GOL1) with yellow carbon atoms; hydrogen bonds are shown as red dotted lines. (B) The α9-α10 loop of a neighboring protomer (chain A, C and D) protrudes towards the central cavity ('inward' conformation), interacting with glycerol (GOL2) in chains A and D. In contrast, chain B of the neighboring α9-α10 loop has shifted away from the cavity ('outward' conformation, purple).</figcaption></figure>

**Tjaard Pijning:** Formal analysis, Methodology, Visualization, Writing – original draft, Writing – review & editing. **Régis Fauré:** Investigation, Writing – review & editing. **Edita Jurak:** Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing.

**Declaration of Competing Interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgements**

We thank the staff at beamline ID23-1 of the ESRF for assistance with X-ray diffraction data collection and Prof. Nicolas Terrapon for providing all sequences of GH154 and performing the CD-HIT [44] analysis.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.nbt.2023.12.011.

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