*2.2. Active Center of Arth*β*DG and Arth*β*DG\_E441Q Mutein*

Catalytic site of *Arth*βDG, located at the bottom of a relatively wide funnel on the top of catalytic Domain 3, is complemented with parts of the chain from Domain 1 and Domain 5 at the entrance of the active cavity (Figure 2). The funnel leading to the active site has a strongly acidic character, which is beneficial for binding of carbohydrate substrates (Figure 3).

**Figure 2.** The dimer of *Arth*βDG\_E441Q and the zoom of one of the active site cavities with lactose.

**Figure 3.** The surface potential visualization at the active site of *Arth*βDG (**A**), *Arth*βDG\_E441Q (**B**), *Ecol*βDG (**C**), and *Klyv*βDG (**D**).

The active site cavity has an acidic character throughout, which facilitates the binding of the saccharide substrate, which is typically lactose. Such a shape of the active site cavity is observed for other βDGs with transglycosylation activities: *Ecol*βDG and *Klyv*βDG [4,7,10] as it facilitates the binding of a larger acceptor of galactosyl group, such as galactose, fructose, or salicin. *Arth*βDG forms a widely open entrance to its catalytic site, which makes it more accessible for the substrate but also promotes product dissociation. Both, easier product dissociation and active sites not being shielded or restricted from solvent may be considered as structural cold-adaptation as it influences the enzymes' turnover rate.

#### *2.3. Structural Analysis of Reactions' Mechanism Catalyzed by Arth*β*DG*

All determined crystal structures showed precisely the changes in the active site of the enzyme in different stages of catalyzed reaction. Visualizing these structural changes helps to understand and explain a classical Koshland double-displacement mechanism occurring during hydrolysis of (1,4)-β-O-glycosidic bond catalyzed by *Arth*βDG (Figure 4).

**Figure 4.** The reaction mechanism of Koshland double displacement with the catalytic residues numbered as for *Arth*βDG [33].

Determined crystal structures of *Arth*βDG complexes with specific ligands may be divided into three groups: complexes with substrates and their analogues (early complexes) *Arth*βDG\_E441Q/LACs, *Arth*βDG/IPTG; the second group are intermediates complexes *Arth*βDG\_E441Q/LACd *Arth*βDG/ONPG; and the third one is the complex with product *Arth*βDG/GAL.

The early complexes with substrate show LAC and IPTG binding in shallow mode, intermediate complexes depicts deep binding of substrate that directly precedes formation of galactosyl-enzyme covalent bond, and the product complex allows description of the product release process.

At the early stage of the reaction, the substrate is bound in the shallow binding site where the galactosyl group is stabilized by a number of H-bonds between its hydroxyl groups and residues N110, E441, E517, M481, H520, and H368 via water molecules and by an interaction with a sodium ion. Additionally, the glucosyl moiety of lactose is stabilized by H395 and E398 via a water molecule, even

though there is already an interaction between substrate and catalytic residues (E441 and E517), and the position of the substrate does not allow access to O-glycosidic bond (Figure 5).

**Figure 5.** Early complexes of *Arth*βDG with saccharide substrate and substrate analogue: the molecule of lactose (**A**) and IPTG (**B**) bound at shallow binding site.

The insertion of substrate into deep binding is associated with movement of F581 phenyl ring, which rotates around Cα-Cβ bond causing shift of aromatic ring by 2.9 Å in the direction of the active center, reducing the volume of the shallow binding site (Figure 6). Surprisingly, no movement of backbone is observed during the transfer of ligand from shallow to deep binding site. During substrate transfer into the deep binding site, the galactosyl moiety, properly positioned in shallow binding stage, is moved deeper into the active site by approximately 2.4 Å being at the same time rotated around an axis perpendicular to the sugar ring by approximately 60◦.

**Figure 6.** Enzyme active site of shallow and deep binding of lactose. Electron density 2Fo-Fc map of lactose in deep (**A**) and shallow (**B**) binding mode (contoured at 1σ). Superposition of enzyme active site in both structures (**C**).

In the deep binding site of *Arth*βDG, the galactosyl ring is stabilized by direct interactions of its hydroxyl groups with E441 (Q441 in mutein), E517, H368, D207, sodium ion and N440, D584, H520 (latter observed by deeper bound lactose in complex of *Arth*βDG\_E441Q\_LACd). Now in *Arth*βDG\_E441Q\_LACd, the NH2 from the amide group of Q441 interacts directly with oxygen from the glycosidic bond of lactose, which is 2.6Å away (Figure 7A). In *Arth*βDG/ONPG, the carboxyl group of E441 interacts directly with oxygen from the glycosidic bond of ONPG, which is at a distance of 2.8 Å (Figure 7B).

**Figure 7.** Late complexes of *Arth*βDG with substrates: the molecules of lactose (**A**) and ONPG (**B**).

The superposition of active sites of *Arth*βDG with *Ecol*βDG shows the conservation of amino acids involved in stabilization of the galactosyl moiety. However, these two enzymes differ in the stabilization of a second moiety of β-d-galactoside (Figure 8). If we consider binding of the natural substrate, lactose, the second moiety is glucopyranose. In the active site of *Ecol*βDG, the glucopyranose ring is stabilized by π-stacking interaction with W999. However, in the *Arth*βDG active site, W999 is substituted by C985. This cysteine residue does not influence stabilization of substrate in shallow binding mode. However, when lactose is bound in deep mode, the center of the glucopyranose ring is at a distance of 4.4 Å from C985 making π-sulphur interaction possible. Thus, substitution of W999 with C985 reduces stabilization of the second moiety of β-d-galactoside during shallow binding mode, but is still creating stabilizing interactions when the substrate is bound in deep mode. Furthermore, this results in creating more space in the close vicinity of the active site by substituting the bulky indol group with a smaller cysteine residue side chain.

**Figure 8.** Superposition of catalytic sites of *Arth*βDG with lactose bound in deep mode (green) and *Ecol*βDG (purple).

After the hydrolysis reaction is completed, the F581 side chain moves back into its previous position, opening the way for galactose molecule evacuation from the active site (Figure 9).

The product, now in half-chair conformation, is still stabilized by a number of interactions: D207, H368, N440, E441, Y482, E517, H520, and C985 (Figure 7). The 'open' position of F581 is also observed for unliganded structures of *Arth*βDG (PDB IDs: 6ETZ) and its mutant *Arth*βDG\_E441Q, suggesting that its movement is dependent upon substrate presence at the shallow binding site.

**Figure 9.** Complex structure of *Arth*βDG with galactose in half-chair conformation bound in the active center.

#### **3. Discussion**

GH2 family β-d-galactosidases are sugar configuration-retaining enzymes that follow a classical Koshland double-displacement mechanism. These crystal structures of *Arth*βDG complexes with ligands enabled characterization of the active site and determined which residues take part in two modes of substrate binding: deep and shallow.

The large rotation of the galactosyl residue during deep binding would most probably result in forming π-stacking interaction with W548. Such a form of intermediate stabilization was described for *Ecol*βDG [7]. It should be noted that a tryptophan residue is the preferred aromatic amino acid for binding of carbohydrates [34,35] and is frequently present in carbohydrate binding domains of proteins. In the case of *Arth*βDG, only one tryptophan, W548, is located in the bottom of the active site. Additionally, three tryptophan residues are present at the entrance to the catalytic pocket (W402, W470, and W773), where they may form platforms for initial sugar binding. Another amino acid which is considered to play an important role in carbohydrate binding is histidine. The active site of *Arth*βDG contains several histidine residues: H334, H368, H395, H520, and H553. Among them, H368 and H520 are directly involved in stabilizing the galactosyl moiety. H520 is primarily involved in stabilization of hydroxyl group O6 of the galactosyl moiety during shallow binding of substrate. When the substrate is moved deeper into catalytic site, H368 stabilizes the position of hydroxyl group O3. It must be noted that the catalytic site architecture of *Arth*βDG is composed such a way that only a sugar moiety with a proper conformation of hydroxyls O2, O3, and O4 can be effectively bound in the active site. Hence, residues forming H-bonds with hydroxyl groups in these positions H368, N440, and D207, play a crucial role in enzyme's specificity.

It is worth noting that a typical chair conformation of the galactosyl ring in substrate (1*C*4) is changing to a half-chair (3*H*4) hkkkkHhhh conformation in the still bound product of the half-reaction. There are many conformations of pyranose ring possible in solution; however, some of them are more stable than others. In the case of lactose, it usually has a relaxed chair conformation in solution. The double displacement mechanism, in which lactose is hydrolyzed by retaining galactosidases, such as *Arth*βDG, undergoes formation of two oxocarbenium ion-like transition states (Figure 4). Such transition states must be formed with sp<sup>2</sup> hybridization and formation of a positive charge on anomeric carbon atom of the substrate. Only a few conformations of galactosyl moiety allow sp2 hybridization on anomeric carbon, one of which is half-chair conformation <sup>3</sup>*H*4, observed for the galactose bound in active site of *Arth*βDG [36].

The rotation of F581 (F601 in *Ecol*βDG) was described as one of the factors associated with the deep binding mode, together with 10 Å movement of a 10-aa loop from Domain 5. However, in the case of *Arth*βDG, the movement of F581 and D207 are the only conformational changes accompanying the reaction mechanism. In fact, the 10-aa loop in *Arth*βDG is stabilized by a number of strong interactions with other parts of Domain 5, in a position allowing better access of the substrate to the active site. It should be noted that it is one of the regions of *Arth*βDG in which the backbone differed significantly from homologous structures. These facts lead us to consider this permanent exposure of the entrance to the active site as a structural adaptation towards activity in cold conditions. Fewer structural hindrances for substrate entering and product leaving the active site can result in a higher turnover rate. Analysis of these obtained crystal structures shows that *Arth*βGD forms a widely open entrance to its catalytic site, which makes it more accessible for the saccharide substrate and promotes product dissociation.

Both galactosyl binding sites, shallow and deep ones, form a net of H-bonds that stabilize this part of the substrate. On the other hand, the glucosyl moiety of lactose, or the non-galactose moieties of IPTG and ONPG, are hardly stabilized by any interactions during shallow binding. The *Ecol*βDG W999 is substituted at *Arth*βDG with a cysteine residue which may stabilize the second sugar ring of the substrate by π-sulphur interactions during deep binding, however such a substitution would render the enzyme less specific toward binding a sugar moiety at this position. Thus, not only disaccharides, but also other galactosides are processed by *Arth*βDG. The enzyme's lack of preference for the second moiety in galactoside may be the main reason for its ability to hydrolyze a wide variety of substrates, as well as for its ability to transfer galactosyl group to a variety of acceptors [16] resulting in an interesting range of potentially useful heterooligosaccharides.

#### **4. Materials and Method**

#### *4.1. Site-Directed Mutagenesis of Gene Encoding Arth*β*DG*

The gene encoding the *Arth*βDG enzyme, which was previously cloned into the pBAD/Myc-His A expression vector [16], has been mutated in a site-specific manner using the Q5 Site-Directed Mutagenesis Kit (NEB, Ipswich, MA, USA) following the manufacturer's protocol. For this purpose, a pair of mutagenic primers was designed and synthesized (Genomed, Warszawa, Poland). Primer ForBglAr32cBm441: 5 GTCCCTGGGCAACCAGGCGGCACCGG3 and primer RevBglAr32m441: 5 CACATGACCACCGAGGCGTGGTTCTTGTCGCGC3 allowed us to introduce a point mutation at 1321 nucleotide position in the gene substituting G with C resulting in the substitution of glutamic acid (E) residue with glutamate (Q) residue in the 441 position of the amino acid chain of *Arth*βDG. Hence, the product of mutated gene expression has been called *Arth*βDG\_E441Q. In theory, this amino acid change should abolish β-d-galactosidase activity of mutein *Arth*βDG\_E441Q.

PCR cycling conditions were as follows: (1) Initial DNA denaturation at 98 ◦C for 30 s; then (2) 25 cycles of PCR product amplification consisting of 10 s of DNA denaturation at 98 ◦C, 20 s of mutagenic primers annealing at 70 ◦C, and 3 min 20 s of PCR product extension at 72 ◦C; and (3) the final PCR product extension at 72 ◦C for 7 min. After PCR, the amplified DNA product was directly added to unique Kinase-Ligase-DpnI (KLD) enzymes mix. Then the product of KLD reaction (5 min at room temperature) was directly used to transform NEB 5-alpha chemically competent

*E.coli* cells (the *lacZ* deletion mutant, Δ (lacZ) M15). After that, transformants were spread on Luria–Bertani agar plates (10 g L−<sup>1</sup> of peptone K,5gL−<sup>1</sup> of yeast extract, 10 g L−<sup>1</sup> of NaCl, and 15 g L−<sup>1</sup> of agar) supplemented with ampicillin (100 μg mL−1), X-Gal (40 μg mL−1) and l-arabinose (200 μg mL<sup>−</sup>1). After plate incubation—firstly at 37 ◦C for 12 h, and then at 22 ◦C for next 12 h—a few recombinant colonies without β-d-galactosidase activity were chosen for further studies. Plasmids isolated using the ExtractMe Plasmid DNA Kit (Blirt, Gdansk, Poland) from selected recombinants were sequenced (Genomed, Warszawa, Poland) and analyzed (blast2go on-line tool). Recombinant plasmid pBAD-Bgal32cB\_E441Q(A) harboring the properly mutated *Arthrobacter* sp. 32cB β-d-galactosidase gene under the control of the PBAD promoter was used for effective production of *Arth*βDG\_E441Q mutein in *E. coli* host [16].

#### *4.2. Expression and Purification of Arth*β*DG and Arth*β*DG\_E441Q*

Heterologous expressions of recombinant *Arth*βDG and *Arth*βDG\_E441Q proteins were performed in the *E. coli* LMG 194 cells transformed with pBAD-Bgal32cB and pBAD-Bgal32cB\_E441Q plasmids, respectively, as previously described. [25] Both proteins were purified by two ion-exchange chromatography steps (weak anion exchanger and strong anion exchanger), followed by a size-exclusion chromatography step.

The fractions containing *Arth*βDG were identified by SDS-PAGE electrophoresis run on 10% SDS-polyacrylamide gel and by enzymatic activity assay with ONPG as a substrate [25], whereas the fractions containing *Arth*βDG\_E441Q were identified by SDS-PAGE only, due to lack of enzymatic activity. The sample buffer was changed into 0.05 M HEPES pH 7.0 and the samples were concentrated using 50 kDa cut-off membrane Vivaspin filters (Sartorius, Goettingen, Germany) up to the protein concentration of 15 mg/mL.

### *4.3. Arth*β*DG Crystallization and Di*ff*raction Data Collection*

Crystals of *Arth*βDG and *Arth*βDG\_E441Q mutein were grown using the same optimization matrix of 25–45% TacsimateTM and pH ranges between 6.0–8.0. All the drops were set up using a seed stock prepared from crystals of *Arth*βDG grown at 35% TacsimateTM pH 7.0 and diluted 10,000 times. Numerous attempts of co-crystallization with ligands were undertaken but no crystal structures of desired complexes were obtained. Furthermore, addition of natural substrate, lactose, prevented formation of *Arth*βDG\_E441Q crystals even at very low concentration of added ligand. Crystal structures of investigated *Arth*βDG and *Arth*βDG\_E441Q complexes were obtained by soaking of native and mutant crystals with desired ligand or ligands mixture. Soaking was performed by adding powder of ligand directly to the crystallization drop. The soaking experiments were performed for 15 min, 30 min, 1 h, 2 h, 6 h, 14 h, and 24 h prior to flash-freezing. The crystals, prior to mounting and flash-freezing, were protected with 60% TacsimateTM of pH corresponding to crystallization conditions [37].

High-resolution diffraction data were collected using synchrotron sources on beamlines 14.1 and 14.2 at BESSY, Berlin, Germany and P13 beamline at PETRA, DESY Hamburg, Germany. The diffraction images were collected with fine slicing 0.1◦ and diffraction data were processed using XDSapp [38]. Crystal structures were solved and refined using the PHENIX program suite [39]. As a model, the structure of *Arth*βDG (PDB ID: 6ETZ) was used.

**Author Contributions:** M.R. performed crystallization; M.R. and A.B. performed synchrotron diffraction data collection, processing, structure solving, and carried out structural analysis; M.R. purified enzyme, refined the structures; M.R. and A.B. prepared the manuscript; M.W. performed native *Arth*βDG enzyme expression in *E. coli* and determined the purification protocol; H.C. designed and performed site-direct mutagenesis experiment resulted in a gene encoding *Arth*βDG\_E441Q mutein; A.W.-W. performed *Arth*βDG-E441Q mutein expression in *E.coli*; A.B. coordinated the project.

**Funding:** This research was funded by National Science Centre of Poland grant number 2016/21/B/ST5/00555 (A.B.) and 2018/28/T/ST5/00233 scholarship (M.R.).

**Acknowledgments:** We thank HZB for the allocation of synchrotron radiation beamtime at BL 14.1, BL 14.2, and PETRA synchrotron at P13.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
