**1. Introduction**

α-D-Galactosidases (EC 3.2.1.22) catalyze the hydrolysis of the nonreducing terminal α-D-galactose (Gal) from α-D-galactosides, galactooligosaccharides, and polysaccharides, such as galactomannans, galactolipids, and glycoproteins. According to the classification of carbohydrate-active enzymes (CAZy) [1], α-D-galactosidases mostly belong to 27, 36, and 110 families of glycoside hydrolases

(GH). They are found also in the GH 4, GH 57, and GH 97 families. The GH 27 and GH 36 enzymes, with a common mechanism of catalysis, have the protein structural (β/α)8-barrel fold in the catalytic domain and similar topology of their active centers, typical for a clan GH D [2]. The GH 27 and GH 36 family members are classical retaining glycoside hydrolyses in accordance with Koshland's classification [3]. These enzymes catalyze the hydrolysis of O-glycosidic bonds by a double displacement mechanism through the galactosyl-enzyme covalent intermediate, as well as the transglycosylation reaction under the specific conditions [4].

α-D-Galactosidases are widespread among terrestrial plants, animals, and microorganisms. These enzymes have found many practical uses in different fields from biomedicine to enzymatic synthesis [4]. The enzymes occur frequently in marine bacteria, especially in γ-Proteobacteria and Bacteroidetes [5–9]. Currently, the genes encoding these enzymes can be found in the genomes of marine bacteria, annotated in the National Center for Biotechnology Information NCBI database. For the first time, α-PsGal was isolated from a cold-adapted marine bacterium *Pseudoalteromonas* sp. strain KMM 701 inhabiting in the cold water in the Sea of Okhotsk. The enzyme attracted our attention due to its ability to reduce the serological activity of B red blood cells. The marine bacterium's α-PsGal was more efficient in the model of B-erythrocyte antigen, than a well-known α-D-galactosidase from green coffee beans, which was usually used in experiments on transformation of donor blood erythrocytes for intravenous injection [10]. The enzyme also interrupted the adhesion of *Corynebacterium diphtheria* to buccal epithelium cells at neutral pH values [11], as well as stimulated the growth of biofilms of some bacteria [12]. These properties of the enzyme determined the possible directions for its practical application in biomedicine. According to the structural CAZy classification, α-PsGal belongs to the GH 36 family [11]. The enzyme is a retaining glycoside hydrolase [13], cleaving the terminal Gal from melibiose Gal-α(1→6)-Glc, raffinose Gal-α(1→6)-Glc-β(1→4)-(Fru), digalactoside Gal-α(1→3)-Gal, and B-trisaccharide Gal-α(1→3)-(Fuc-α(1→2)-Gal) [10]. However, the most important glycosynthase properties of the enzyme have yet to be studied.

The present article aimed to compare the properties of recombinant α-D-galactosidase from marine bacterium *Pseudoalteromonas* sp. KMM 701 (α-PsGal) and its mutants, where the predicted functionally important residues D451 and C494 of the active center were replaced by the less reactive alanine (A) and asparagine (N) residues, respectively. Major attention was focused on the regioselectivity of the transglycosylation reaction.

#### **2. Results**

#### *2.1. Bioinformatics Analysis and Homology Modeling of α-PsGal Protein 3D-Structure*

Bioinformatics analysis and homology modeling of the protein structure was completed to elucidate the amino acid residues roles of α-PsGal for catalysis. The results of homology modeling of the α-PsGal protein three-dimensional (3D) structure are shown in Figure 1.

The homology model of the α-PsGal 3D-structure was constructed by the package Molecular Operating Environment version 2018.01 (MOE) [14] using the crystal structure of α-galactosidase from *Lactobacillus acidophilus* of the GH 36 family [15] as a template (Figure 1a). The amino acid sequence of α-galactosidase from the marine bacterium has 28.7% identity and 44% similarity with the sequence of the prototype. The superposition (root mean squared difference (RMSD) of the Cα-atoms = 0.8 Å) of the α-PsGal homology model with the template active site revealed that the D451 and C494 residues superimposed on the nucleophile/base D482 and the substrate binding C530 residues in the template, respectively (Figure 1b) [15]. Thus, the predicted structure of α-PsGal is applicable for in silico mutagenesis and molecular docking studies. The evidence from Figure 1c suggests that C494 takes part in forming a network of hydrogen bonds between the catalytic residues D451, D516, and the hydrolysis product D-galactose (D-Gal). To eliminate the roles of D451 and C494 residues, they were substituted by A451 and N494, respectively.

**Figure 1.** Homology model of α-PsGal three-dimensional (3D) structure generated using X-ray structure of the α-galactosidase of *Lactobacillus acidophilus* (PDB ID 2XN2) as a template: (**a**) 3D-model of α-PsGal structure in a ribbon diagram representation: α-helixes (red), β-strands (yellow), coils (white), and turns (blue); (**b**) superimposition of the α-PsGal homology model (orange) with template active sites (turquoise); D-galactose is shown by sticks (green); and (**c**) the binding site of D-galactose in the active center of α-PsGal homology model. Hydrogen-bond contacts were determined using the Protein Contacts module of Molecular Operating Environment version 2018.01 (MOE) program (Chemical Computing Group ULC: 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2018) and are shown with a dashed line.

#### *2.2. Enzyme Production and Purification*

The recombinant wild α-PsGal and its mutants D451A and C494N were expressed and purified successfully as soluble proteins with 97% purity according to the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) data. The gel electorophoregrams of the enzyme preparations were obtained in different experiments and are summarized in Figure 2.

**Figure 2.** SDS-PAGE (12.5%) of the recombinant wild α-PsGal (lanes 1 and 2 before and after final purification stage, respectively) and its D451A and C494N mutants after the final stages of purification (lanes 3 and 4, respectively); molecular weight markers are shown in lane 5.

The molecular weight of the protein fraction of the cellular extract *Escherichia coli* Rosetta (DE3)/40Gal (Figure 2, lane 1) corresponds to the chimeric recombinant α-PsGal fused with the plasmid pET-40 b (+) chaperone protein DsbC overhang (80 kDa (α-PsGal) + 32.5 kDa (DsbC) = 112.5 kDa). After the final stage of purification and treatment with enterokinase, the molecular weight of mature recombinant proteins α-PsGal, D451A, and C494N were ca. 80 kDa (Figure 2, lanes 2–4, respectively).

### *2.3. Properties of Recombinant Wild-Type α-PsGal and Mutant C494N*

The mutant D451A did not exhibit any hydrolytic activity against either melibiose or pNP-α-Gal, indicating the extreme importance of this residue for the functioning of this enzyme. The specific activities of the recombinant wild α-PsGal and mutant C494N, with the use of pNP-α-Gal as a substrate, were 90.0 and 0.87 U/mg, respectively. Further comparative studies showed some similarities and differences in the enzymatic properties of the wild recombinant α-PsGal and its mutant C494N.

#### 2.3.1. Circular Dichroism Spectra of Wild-Type α-PsGal and Mutant C494N

To identify the similarity of the secondary structure of the wild α-PsGal and C494N mutant; circular dichroism (CD) spectroscopy was used (Figure 3).

**Figure 3.** Circular dichroism spectra of wild α-PsGal (1) and C494N mutant (2) with 0.1 M sodium phosphate buffer (pH 7.0), 25 ◦C, and 0.1 cm cell.

The CD spectra of the wild α-PsGal and C494N mutant were approximately identical binshape and amplitude of the bands (Figure 3, spectrum 1 and 2, respectively). Calculation of the secondary protein structure elements (see p. 4.4.1.) indicated the presence of 27.9% and 27.9% α-helices, 19.9% and 21.4% β-structures, and 28.9% and 28.9% disordered structure, including 23.3% and 21.8% β-turns for the wild α-PsGal and C494N mutant, respectively. The determination of the tertiary structure

class using the same software package established that the wild α-PsGal and C494N mutant belong to α + β tertiary structure class of proteins. Thus, the C494N point mutation in the active site does not significantly affect the secondary structure of the enzyme as a whole.

2.3.2. Effect of pH on Wild-Type α-PsGal and C494N Mutant Activities

It is evident from Figure 4 that the wild α-PsGal (Figure 4a) retains activity in a wide pH range (6.5–8.0).

**Figure 4.** Effect of pH on the activity of enzymes: (**a**) wild-type α-PsGal and (**b**) mutant C494N. Fragments of curves correspond to 0.1 M sodium citrate buffer (1), 0.1 M sodium phosphate buffer (2), and 0.1 M Tris HCl buffer (3).

Citrate and phosphate anions were preferable for enzyme activity. The tris ion was an effective inhibitor of the activity of wild α-PsGal and mutant C494N. The replacement of cysteine 494 with asparagine residue did not significantly change the pH effect on the enzyme activity (Figure 4).

#### 2.3.3. Effect of Temperature on Wild-Type α-PsGal and Mutant C494N Activities

It is evident that wild α-PsGal is a cold-active enzyme due to retaining about 30% of its activity at 5 ◦C (Figure 5a). The activity of the mutant C494N reached a maximum at higher temperature values than wild α-PsGal (Figure 5b).

**Figure 5.** Effect of temperature on activity of enzymes: (**a**) the dependence of relative activity on temperature of wild α-PsGal (1) and C494N mutant (2) and (**b**) thermal stability of wild α-PsGal (1) and C494N mutant (2). The solid line (3) indicates 100% activity and the dashed line (4) indicates 50% activity.

Although the middle of the temperature transition lay at the same temperature of ~32 ◦C, the temperature inactivation in the wild α-PsGal started at lower temperatures than in the C494N mutant. Both the recombinant wild α-PsGal and the C494N mutant proved to be more thermostable enzymes than the natural α-galactosidase from the marine bacterium *Pseudoalteromonas* sp. KMM 701 [10].

2.3.4. Kinetic Parameters of Catalytic Reactions for Wild-Type α-PsGal and Mutant C494N

Michaelis-Menten constant (*K*m) and maximal rate (*V*max) of pNP-α-Gal hydrolysis were defined from Lineweaver-Burk graphs.

Catalytic parameters of the reaction are summarized in Table 1.


**Table 1.** Catalytic properties of wild α-PsGal and mutant C494N.

Conditions of reactions: 0.05 M sodium phosphate, pH 7.0, 20 ◦C; concentration of α-PsGal: 34 μ/mL, mutant C494N: 22 μ/mL.

It is evident from Table 1 that the replacement of cysteine 494 in the active site of α-PsGal with asparagine residue leads to an approximately 200-fold decrease in the efficiency (*k*cat/*K*m) of the enzyme retaining the identical affinity (*K*m) of both enzymes to the standard substrate pNP-α-Gal. This indicates the extreme importance of C494 in the manifestation of α-PsGal activity (Table 1).

#### 2.3.5. Theoretical Model of the D-Gal Complexes with Wild α-PsGal and Mutant C494N

Figure 6 shows two-dimensional (2D) diagrams of the D-Gal complexes with the active center of the wild α-PsGal (Figure 6a) and mutant C494N (Figure 6b) built by molecular docking in the MOE program.

**Figure 6.** Two-dimensional (2D) diagrams of the D-Gal binding sites in (**a**) wild α-PsGal and (**b**) mutant C494N.

In silico analysis of the wild α-PsGal-D-Gal and mutant C494N-D-Gal complexes (Figure 6) showed that substitution C494N led to the emergence of new hydrogen bonds (Figure 6b) and to the

increase in binding energy of the reaction product D-Gal in the binding site of the enzyme (Table S1). This probably reflected a decrease in the values of the *k*cat and *k*cat/*K*m constants of the enzyme.
