*2.5. Production of Transglysolylation Reactions*

The transglycosylation properties of the purified wild α-PsGal, mutant D451A, and C494N were tested using melibiose (1.10 mmol/mL) and pNP-α-Gal (0.25 mmol/mL) as substrates at pH 7.0 and 8 ◦C and 20 ◦C. The concentration of the substrates significantly exceeded the enzyme concentration. The composition of incubation mixtures and units of the enzymes are shown in Table 3.

**Table 3.** Composition of reaction mixtures for monitoring of transglycosylation with various forms of the recombinant α-galactosidase from the marine bacterium *Pseudoalteromonas* sp. KMM 701.


Since the activity of mutant C494N was much lower compared with the wild type (Table 3), the reaction mixtures containing weakly active C494N (0.012 U) and wild-type α-PsGal with an activity of 0.015 U were incubated for seven days. In order to avoid the thermal inactivation of the enzymes, the reactions were carried out in a refrigerator at 8 ◦C. Therefore, the products from the reactions at low (8 ◦C) and moderate (20 ◦C) temperatures were compared. The conditions of rescue experiments and the investigation of glycosynthase properties of the mutant D451A in the presence of sodium azide, as an external nucleophile, are also shown in Table 3.

#### 2.5.1. Thin Layer Chromatography (TLC)

The results of TLC analyses of the reaction mixtures after the action of recombinant α-PsGal, as well as D451A and C494N mutants, on melibiose (Gal-α(1→6)-Glc), and pNP-α-Gal were obtained under different conditions (Figure 7).

**Figure 7.** Thin layer chromatography (TLC) profiles of the hydrolysis and transglycosylation products produced by recombinant α-PsGal. Lanes 1 and 8—α-PsGal with Gal-α(1→6)-Glcα,β at 20 ◦C and 8 ◦C, respectively. Lanes 2 and 4—α-PsGal with pNP-α-Gal at 20 ◦C and 8 ◦C, respectively. Lane 3—α-PsGal with mixture of Gal-α(1→6)-Glcα,β/pNP-α-Gal at 20 ◦C. Lane 5—D451A mutant with Gal-α(1→6)-Glcα,β/NaN3. Lane 6—D451A mutant with pNP-α-Gal/NaN3. Lane 7—D451A mutant with pNP-α-Gal. Blank mixtures: lane 9—Gal-α(1→6)-Glcα,β, lane 10—pNP-α-Gal, lane 11—Gal, and lane 12—Glc.

Lanes 1–8 correspond to each experiment. The melibiose, pNP-α-Gal, galactose (Gal), and glucose (Glc) standards (Figure 7, lanes 9–12, respectively) spots on the chromatogram had retardation factors (Rf) of 0.39, 0.83, 0.52, and 0.56, respectively. The conditions of reactions are listed in Table 3.

After separation of the reaction mixtures with the melibiose processed by the recombinant α-PsGal at 20 ◦C and 8 ◦C (Figure 7; lanes 1 and 8; respectively), two new spots with Rf 0.35 and 0.26 occurred in the chromatogram; in addition to the hydrolysis products Gal and Glc (Rf 0.52 and 0.55; respectively). These new sugars could be interpreted as transglycosylation products. In the mixture of the reaction products of pNP-α-Gal treated with the recombinant α-PsGal at 20 ◦C and 8 ◦C; the spots with Rf 0.61 and 0.34, corresponding to new sugars along with the spots of pNP-α-Gal and Gal (Rf = 0.82 and 0.52; respectively), were observed (Figure 7; lanes 2 and 4; respectively). It is interesting to note that the compound with the Rf value of 0.34 was not formed by the action of the recombinant wild α-PsGal on the mixture of melibiose (donor)/pNP-α-Gal (acceptor) (Figure 7; lane 3). The D451A mutant showed no activity toward pNP-α-Gal (Figure 7; lane 7). Unfortunately, reactivation experiments with external nucleophile sodium azide failed to restore the activity of the nucleophile mutant enzyme (data not shown). However, in the presence of sodium azide (Figure 7; lanes 5 and 6), the traces of unidentified compounds (Rf value of 0.42 and 0.86; respectively) were formed under an action of D451A on melibiose and pNP-α-Gal

#### 2.5.2. MALDI Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were recorded for five samples (S1): α-PsGal with a mixture of melibiose and pNP-α-Gal, α-PsGal with pNP-α-Gal, D451A with pNP-α-Gal and NaN3, C494N with pNP-α-Gal, as well as standard mixtures: melibiose, pNP-α-Gal, Gal, and Glc. The molecular weights of sugars (both products of hydrolysis and substrates) were registered as sodium adducts [M + Na]<sup>+</sup> in positive-ion mode, where M represents the neutral molecule: [Hex + Na]+ at *m*/*z* 203.06, [Hex2 + Na]<sup>+</sup> at *m*/*z* 365, [pNP-Hex2 + Na]<sup>+</sup> at *m*/*z* 486, and [Hex3 + Na]<sup>+</sup> at *m*/*z* 527. pNP-α-Gal was found as [pNP-α-Gal + Na]+ ion at *m*/*z* 324. MALDI mass spectra showed only the semiqualitative composition of the transglycosylation products (Table 4).



According to the mass spectral data, the signal of the [Hex2 + Na]<sup>+</sup> ion at *m*/*z* 365 of disaccharide (Hex2) was major in the MALDI MS of the reaction mixture of α-PsGal with melibiose/pNP-α-Gal. In addition, there were signals of the new [pNP-Hex2 + Na]<sup>+</sup> and [Hex + Na]<sup>+</sup> ions in the spectrum (Figure S1a) at *m*/*z* 486 and 203, respectively. Along with signals of the hydrolysis product [Hex + Na]<sup>+</sup> ion at *m*/*z* 203 and the remainder of the substrate [pNP-Hex + Na]+ ion at *m*/*z* 324, new signals of [Hex2 + Na]<sup>+</sup> and [pNP-Hex2 + Na]<sup>+</sup> ions were observed in the matrix-assisted laser desorption ionization-mass spectroscopy (MALDI-MS) of the reaction mixture of the wild α-PsGal with pNP-α-Gal at 8 ◦C (Figure S1b). The signals of new carbon compounds were not detected in the MALDI-MS of the product mixture obtained under the action of mutant D451A on pNP-α-Gal in the presence of NaN3 (Figure S1c). The main signal of the [Hex2 + Na]+ ion at *m*/*z* 365 was identified in the spectrum of the reaction mixture of the C494N mutant with pNP-α-Gal. The spectrum also contained two minor signals of [pNP-Hex2 + Na]+ at *m*/*z* 486 and [Hex + Na]<sup>+</sup> ion at *m*/*z* 203 (Figure S1d).

To identify the regioselectivity of the transglycosylation, we used tandem electrospray ionization mass spectrometry (EISMS/MS) with collisional induced dissociation (CID) in positive ion mode. The EISMS profiles of the reaction products are illustrated in Figures S2, S3, and S4. The linkage identifications were based on the fragmentation rules described earlier [16] for negative ion mode, and further supported by positive ion mode [17]. In brief, the absence of fragment ions from cross-ring cleavages in a disaccharide suggests a 1,3-type linkage, the 0,2A2-type fragment ion suggests 1,4-type linkages, and both 0,2A2 and 0,3A2-type ions suggest 1,6-type linkages in disaccharides. The nomenclature for the mass spectrometric fragmentation of glycoconjugates was suggested by Domon and Costello [18].

Ion signals of [Hexn + Na]+, *n* = 1–3, at *m*/*z* 203, 365, and 527 were the major components found by EISMS among the reaction products of melibiose and α-PsGal (Figure S2a). The CID ESIMS/MS fragmentation pattern of a trisaccharide ion suggested Hex-(1→4)-Hex-(1→6)-Hex structure (Figure S2b). Fragment ion 0,2A2 at *<sup>m</sup>*/*<sup>z</sup>* 305 and 0,3A2 at *<sup>m</sup>*/*<sup>z</sup>* 275 indicated the presence of both 1→4- and 1→6-*O*-glycosidic links between two hexoses in disaccharide (Figure S2c). The question concerning the presence of the (1→3)-linked hexoses remained unclear, since the disaccharide Galα-(1→3)-Gal could be identified only by the absence of fragment ions from cross-ring cleavages [16]. Ion signals of [Hexn + Na]+, *n* = 1,2, at *m*/*z* 203 and 365 were the major components found by EISMS among the reaction products of melibiose and mutant C494N (Figure S3a). The CID ESIMS/MS fragmentation pattern of a disaccharide ion suggested Hex-(1→4)-Hex only (Figure S3b), so the fragment ion 0,2A2 at *m*/*z* 305 was observed. The type of *O*-glycosidic bond in the pNP-glycosides could not be established because the mobile proton at the glycosyl hydroxyl was blocked. In this case, fragmentation was not observed (Figure S3c–e and Figure S4b) [19].

We used heavy 18O-water for the transglycosylation experiment; we deemed it the most interesting substrate. The use of buffered heavy-oxygen water and liquid chromatography (LC), coupled with ESI-MS/MS (LC-ESIMS/MS) allowed simultaneous observing of the products of hydrolysis and transglycosylation. Since the transfer of heavy 18OH-group produces a +2 mass shift, it was possible to distinguish between fragment ions, retaining the positive charge on the reducing end from the charge on the nonreducing end. Figure 8 shows the kinetics of the consumption and accumulation of the hydrolysis and synthesis products with the use of heavy-oxygen water.

**Figure 8.** Mass spectrometry monitoring of the reaction of melibiose hydrolysis and transglycosylation catalyzed by wild α-*Ps*Gal in the buffered heavy-oxygen water at 20 ◦C. (**a**) Experimental time-dependent changes in integral intensity of matrix-assisted laser desorption ionization-mass spectroscopy (MALDI-MS) signals of the Hex2 ions at 365 *m*/*z* (1) and 367 *m*/*z* (2), Hex at 203 *m*/*z* (3) and 205 *m*/*z* (4), and Hex3 at 527 *m*/*z* (5) and 529 *m*/*z*; (**b**) time dependences of the MALDI-MS signals intensity of Hex3 ions at 527 *m*/*z* (1) and 529 *m*/*z* (2) in an expanded scale.

According to the results (Figure 8), the consumption of Hex2 (Figure 8a, curves 1 and 2) was accompanied by the appearance of Hex (Figure 8a, curves 3 and 4), new Hex2 (Figure 8a, curves 1 and 2), and Hex3 (Figure 8b).

#### 2.5.3. NMR Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the transglycosylation regioselectivity and product structures. To avoid loss of information about minor transglycosylation products, the proton (1H) and carbon (13C) NMR spectra were analyzed without separating the reaction mixtures into individual compounds. The 1H and 13C signals of the anomeric atoms of the substrates and the products were assigned according to the respective reference data [20–22], as well as by H,H-Correlation Spectroscopy (COSY) and Heteronuclear Single-Quantum Correlation (HSQC) experiments. The identified signals are shown in Table 5.


**Table 5.** Anomer signals of nuclear magnetic resonance (NMR) spectra of substrates and products of hydrolysis and transglycosylation catalyzed by wild α-PsGal and its mutant C494.

\* N/D = not defined.

The composition and yields of transglycosylation products in each case are shown in Table 6.

**Table 6.** The yield and structure of the products of transglycosylation reactions catalyzed by the recombinant wild α-PsGal and its mutant C494N based on NMR data.


In the 1H NMR spectrum of the reaction mixture obtained after the action of recombinant α-PsGal on melibiose, the α,β1H signals of galactose (Gal) (**1**) and glucose (Glc) (**2**), which are the hydrolysis products, along with the major α,β1H signal of transglycosylation product Gal-α(1→6)-Galα,β (**3**) and a minor signal of Gal-α(1→4)-Galα,<sup>β</sup> (**4**), were observed (Tables <sup>5</sup> and 6). In the 1H NMR spectrum of the products obtained after the action of recombinant α-PsGal on pNP-α-Gal, the signals of the α1H atoms were registered and characterized for the major autocondensation product Gal-α(1→6)-Gal-α-pNP (**6**) and a minor autocondensation product Gal-α(1→3)-Gal-α-pNP (**7**), as well as for the transglycosylation products Gal-α(1→6)-Galα,β (**3**) and Gal-α(1→4)-Galα,β (**4**) (Tables 5 and 6). The substituted bigalactoside Gal-α(1→6)-Gal-α-pNP (**6**) was a major product of transglycosylation obtained under the action of the recombinant α-PsGal on the mixture of melibiose (donor) and pNP-α-Gal (acceptor), whereas unsubstituted bigalactosides, Gal-α(1→6)-Galα,β (**3**) and Gal-α(1→4)-Galα,β (**4**), were synthesized in small amounts. Signals of α1H of Gal-α(1→6)-Gal-α-pNP (**6**) were found in the 1H NMR spectrum of reaction products observed in the reaction mixture with the C494N mutant with pNP-α-Gal as the substrate. In the last case, there were no α1H signals found for compound Gal-α(1→3)-Gal-α-pNP (**7**). Analysis of the reaction mixture after the action of the D451A mutant on pNP-α-Gal did not reveal new signals in the NMR spectra except for the signals corresponding to pNP-α-Gal.

#### **3. Discussion**

The catalytic properties and structure-function relationships for the marine bacterial α-galactosidase from the GH 36 family, whose genes frequently occur in the genomes of marine bacteria, were characterized for the first time for recombinant α-galactosidase from the marine bacterium *Pseudoalteromonas* sp. KMM 701 (α-PsGal). As a result of our bioinformatic analysis of the amino acid sequence of the enzyme and homologous modeling of the 3D structure, presumably catalytic (D451 and D516) and substrate-binding (C494) residues—extremely important for the functioning of the enzyme—were identified. The predicted nucleophilic residue D451 and substrate-binding residue C494 were replaced with A451 and N494, respectively. Properties of the mutant D451A and C494N were investigated with comparison to wild α-PsGal.

We showed that α-PsGal and its mutant C494N are cold-active enzymes characterized by their neutral pH-optima (6.5–8.0) and low thermostability of 20 to 30 ◦C among the known α-galactosidases. The wild enzyme exhibited about 30% activity at 5 ◦C. No data on temperature and pH effects on the activity were available in the literature for the prototype α-galactosidases from the mesophiles *L. acidophilus.* The α-galactosidases from different mesophilic lactobacilli showed an acidic optimum activity, in the pH range from 5.2 to 5.9, and maximum activity between higher temperatures of 38 to 42 ◦C [23] compared with α-PsGal. The optimal temperature for the activity of the AgaA enzyme from psychrophilic lactic acid bacterium *Carnobacterium piscicola* was 32 to 37 ◦C [24]. The optimum temperature of the enzyme from *Lactobacillus fermentum* was found to be 45 ◦C. The enzyme was inactivated at temperatures higher than 55 ◦C and stable in wide ranges of temperatures and pH [25,26]. As for thermophilic enzymes from bacteria-thermophiles and hyperthermophiles *Bifidobacterium adolescentis* DSM 20083, *B. stearothermophilus*, *Thermus brockianus* [27], *Thermus sp. T2* [28], *Thermoanaerobacterium polysaccharolyticum* [29], and *Thermotoga maritime,* their temperature optimums were 75 to 100 ◦C. The last enzyme was inactive at 30 ◦C [30].

Capability of catalyzing a transglycosylation reaction is an inherent property of all members of the retaining α-D-galactosidases of the GH 27 and GH 36 families [4]. The inverting α-D-galactosidases of the GH 110 family [31], as well as NAD+- and Mn2+-dependent α-D-galactosidases found in the family GH 4 [32], have lost their transglycosylation properties. To date, there is no information about the transglycosylation ability of α-D-galactosidases from the GH 97 and GH 57 families.

It is known that the first step in the catalytic reaction is cleavage of the glycosidic bond of the melibiose or pNP-α-Gal molecules, as well as the formation of the covalent galactosyl-enzyme intermediate. The molecules of Glc and pNP are leaving groups. In the second step, water or some carbohydrate molecules attack the covalent galactosyl-enzyme intermediate, and then hydrolysis or transglycosylation, respectively, can be observed. In the case where the substrate is an attacking molecule, we can observe an autocondensation reaction (Figure S5).

α-PsGal catalyzed synthesis with a total yield of transglycosylation products ranging from 6.0% to 12% (Table 6), similar to the known retaining bacterial galactosidases of the GH 36 family. It was difficult to identify the structures of the transglycosylation products without appropriate standards. However, the use of three methods (TLC, MALDI MS in conjunction with ESIMS/MS, and NMR) provided a suggestion of the relationships between the sugars' molecular weights and the type of O-glycoside bonds in the synthesized oligosaccharides.

TLC is commonly used to analyze low-molecular-weight sugars and their derivatives that differ in the number of carbon atoms, configurations, and molecule sizes. If two carbohydrates have one of these three different characteristics, they can be separated [33]. Based on the results, we assumed that the spot with Rf of 0.35 corresponds to bihexoses, distinguishable from melibiose by the configuration of the stereocenter, but the spot with Rf of 0.26 corresponds to sugars distinct from melibiose by the degree of polymerization (Figure 7, lane 1). The 1H NMR signals of the anomeric atoms of the trisaccharides were not detected. However, the signal of [Hex3 + Na]+ at *m*/*z* 527 was observed in the MALDI MS (Figure S2a). The structure of the trisaccharide Hex-(1→4)-Hex-(1→6)-Hex was established by electrospray ionization tandem mass spectrometry as Gal-(1→4)-Gal-(1→6)-Glc (Figure S2b). The kinetic of accumulation and consumption of Gal-(1→4)-Gal-(1→6)-Glc was registered with the use of heavy-oxygen water (Figure 8b). In the NMR spectrum of the reaction mixture, we found the 1H signals of the anomer atoms (1→6)-α- and (1→4)-α-linked bigalactosides only. In this connection, we think that the spot with an Rf of 0.35 corresponds to two poorly shared (1→6)-α- and (1→4)-α-linked bigalactosides Gal-α(1→6)-Galα,β (**3**) and Gal-α(1→4)-Galα,β (**4**), respectively. This assumption was confirmed by EISMS-MS (Figure S2c).

Thus, when melibiose was used as the substrate, the enzyme synthesized the (1→6)-α-linked bigalactosides (Figure S2c), similar to all known melibiases of the GH 36 family [16,34–42] and to their closely-related GH 27 representatives [43–52]. Furthermore, α-PsGal formed the (1→4)-α-linked bigalactosides as described for mesophilic terrestrial α-D-galactosidases from *Bifidobacterium breve* 203 [35], *Lactobacillus reuteri* [16], and the acidic GH 27 family α-D-galactosidases (AgaBf3S) from the bacterium *Bacteroides fragilis*. The latter was able to transfer galactosyl residues from pNP-α-Gal in lactose Gal-β(1→4)-Glc with the efficiency and strict (1→4)-α-regioselectivity [52], whereas α-PsGal synthesized both (1→6)-α- and (1→4)-α-*O*-glycoside bonds in the bigalactosides from melibiose in the ratio of 9:1 at 20 ◦C and 5:1 at 8 ◦C. It is interesting to note that glucose, which is released from melibiose, did not participate in the transglycosylation reaction as an acceptor because its content in the mixtures was almost half of all products without any change in the course of the reaction (Table 3).

Similarly, we established the structure of the autocondensation products in the mixtures of α-PsGal and pNP-α-Gal (Figure 7, lanes 2 and 4, respectively). α-PsGal was able to produce novel compounds by catalyzing the autocondensation reaction of pNP-α-Gal. Both the substituted Gal2-pNP with (1→6)-α- and (1→3)-α-O-glycoside bonds and unsubstituted Gal2 with (1→6)-α- and (1→4)-α-*O*-glycoside bonds were found in the reaction mixture. The ratio of (1→6)-α-:(1→3)-α-linked Gal2-pNP was 7:1, but the ratio for unsubstituted (1→6)-α-:(1→4)-α-linked bigalactosides was 3:1 at 20 ◦C. The ratio of (1→6)-α-:(1→4)-α-linked bigalactosides reached up to 2:1 at 8 ◦C (Table 6).

The transglycosylation properties are well-studied for the highly thermoresistant GH 36 α-D-galactosidase from the hyperthermophilic bacterium *Thermotoga maritima* (TmGal36A). This enzyme catalyzes an autocondensation reaction with pNP-α-Gal as a substrate, forming substituted (1→2)-α-, (1→3)-α- and (1→6)-α-linked Gal2-pNP [22]. In total, the wild TmGal36A can produce up to 5.5% transglycosylation products. The mechanism of the hydrolysis and synthesis in TmGal36A is not favorable for the formation and breaking of the (1→4)-α-O-glycosidic linkage [22], unlike α-D-galactosidases from human intestine [34–37] and α-PsGal from marine bacterium.

The replacement of the predictive nucleophilic residue D451 to A451 in the active center led to complete loss of the ability of α-PGal to catalyze the hydrolysis. For unknown reasons, the rescue strategy, with an addition of the external nucleophilic sodium azide, proved to be ineffective in this case. Molar concentrations of sodium azide or sodium formate were unable to restore or increase the activity of the mutant D425G of α-D-galactosidase from archaeon *Sulfolobus solfataricus* [53]. Sodium azide did not inhibit any activity of the wild enzyme α-PsGal [10], but it did not restore the activity in its mutant D451A. Galactosyl-β-azide was not found both either of the reaction products of mutant D451A and pNP-α-Gal substrate, as occurred in the experiment with TmGal36A [54]. The D327G mutant of TmGal36A lost hydrolytic properties but retained glycosynthase properties and became an effective α-galactosynthase, which could produce various galactosylated disaccharides from galactosyl-β-azide as a donor and pNP-α(β)-galactosides as acceptors [55].

The mutation C494N changed the specificity for α-PsGal in the synthesis of O-glycoside bonds. Under the action of the C494N mutant on pNP-α-Gal, the yield of pNP-Gal-α-(1→6)-Gal (**6**) decreased, whereas pNP-Gal-(1→3)-α-Gal was not observed. In addition, the content of Gal-(1→4)-α-Gal (**4**) increased two-fold (Table 6). In the literature, it has been reported that the substitution of some bulk residues in the active site of α-D-galactosidase Aga A from *Bacillus stearothermophilus* KVE39 resulted in a 4.5-fold increase in the yield of substituted (1→3)-α-linked compared with pNP-Gal-α-(1→6)-Gal [37]. A number of single and double substitutions of protruded residues in the active site of α-D-galactosidase from *Bifidobacterium adolescentis* DSM 20083 led to an increase in the yield of the total transglycosylation products but they did not change the regioselectivity of the reaction [22,38].

#### **4. Materials and Methods**

#### *4.1. Materials*

The 4-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal), melibiose (Gal-α-(1→6)-Glc), galactose (Gal), glucose (Glc), Bovine serum albumin (BSA), NaN3, and 2,5-dihydroxybenzoic acid were purchased from Sigma Chemical Company (St. Louis, MO, USA). Encyclo DNA-polymerase and enterokinase were purchased from Evrogen (Moscow, Russian Federation). Sodium phosphates, oneand two-substituted, were purchased from PanReac AppliChem GmbH (Darmstadt, Germany). IMAC Ni2+ Sepharose, Q-Sepharose, Mono-Q, and Superdex-200 PG were purchased from GE Healthcare (Uppsala, Sweden). Heavy-oxygen water was purchased from Component Reactive (Moscow, Russia).
