*3.1. Materials*

The 4-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal), D-galactose (Gal), and 4-nitrophenyl-<sup>α</sup>-*N*-acetylgalactosaminide (pNP-α-NAcGal) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Encyclo DNA-polymerase and enterokinase were purchased from Evrogen JSC (16/10 Miklukho-Maklaya str., Moscow, Russian Federation). Nco I and Sal I were purchased

from New England Biolabs (NEB), Ipswich, MA, USA. pET 40 b(+) plasmid was purchased from Invitrogen, Carlsbad, CA, USA. Bacto-tryptone, sorbitol, MgCl2, KH2PO4, kanamycin, glycerin phenyl methanesulfonyl fluoride PMSF were purchased from (Helicon, Moscow, Russian Federation, Kutuzovsky Prospect, 88). Sodium phosphates one- and two-substituted were purchased from PanReac AppliChem GmbH (Ottoweg 4, Darmstadt, Germany). IMAC Ni2+ Sepharose, Q-Sepharose, Mono-Q, and Superdex-200 PG were purchased from GE Healthcare (Uppsala, Sweden). EtOH, trifluoroacetic acid (TFA), CD3OD were from the Russian Federation.

## *3.2. Experimental Equipment*

Optical rotation was measured on Perkin-Elmer 343 polarimeter (Waltham, MA, USA). The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Avance III-700 spectrometer (Bruker BioSpin GmbH, Silberstreifen 4, D-76287 Rheinstetten/KarlsruheIpswich, Germany) at 700 and 175 MHz, respectively. The chemical shifts were correlated in accordance with the CD3OD signals (δH 3.30/δC 4 9.60). Electrospray ionization (ESI) mass spectra (including HRESIMS) were measured using a Bruker Impact II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). HPLC was performed using Shimadzu instrument (Shimadzu Corporation, Kyoto, Japan) with a diffraction refractometer RID-DE14901810 and YMC-ODS-A column (YMC CO., LTD., Kyoto, Japan). Microplate spectrophotometer (BioTek Instruments, Highland Park, Winooski, VT, USA) was used for measuring of optical density at 400 nm (D400).

#### *3.3. Collection and Identification of Sponge Material*

Samples of the sponge *Monanchora pulchra* N 047-028 and N 047-243 were collected by the dredging method during the 47th scientific cruise of the R/V *Akademik Oparin* in August 2015 at the Chirpoi (49◦24.1 N, 154◦17.8 E, depth of 139 m) and Onekotan, the Kuril Islands (49◦24.1 N and 154◦17.8 E, depth of 135 m). Identification of sponges was performed by V.B. Krasokhin. The voucher specimens are kept in the collection of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences (www.piboc.dvo.ru).

#### *3.4. Isolation and Purification of Compounds*

The freshly collected *M. pulchra* samples (N 047-243 and N 047-28) were extracted with EtOH and a part of which (30 mL) was concentrated in vacuo. The residual part was chromatographed on a microcolumn (10 × 12 mm) with YMC\*GEL ODS-A reversed-phase sorbent (75 μm) using aqueous EtOH (40%), and then EtOH (65%)–H2O (35%)–TFA (0.1%). The eluates with TFA were evaporated. The compounds were isolated by HPLC using YMC-ODS-A column (250 × 10 mm) and EtOH (65%)–H2O (35%)–TFA (0.1%) to afford pure compound **1** (4.0 mg) from the sample 047-243, as well as compounds **2** (0.8 mg), and **3** (0.8 mg) from the sample 047-28.

Monanchomycalin B (**1**): high-resolution electrospray ionisation mass spectrometry (HRESI MS) *m/z* 785.6259 [M + H]+, (calcd. for C45 H81 N6O5: 785.6263);

Monanchocidin A (**2**): HRESI MS *m/z* 859.6267, [M + H]+, (calcd. for C47 H83 N6O8: 859.6267); Normonanchocidin A (**3**): HRESI MS *m*/*z* 758.5792, [M + H]+, (calcd. for C43 H76 N5O6: 758.5790).

#### *3.5. Production of Recombinant Enzymes*

#### 3.5.1. Production and Purification of Recombinant α-D-galactosidase

The recombinant wild-type α-D-galactosidase α-PsGal was produced as described earlier [35]. The plasmid DNA pET-40b(+) containing insertion of the gene from the marine bacterium *Pseudoslteromonas* sp. KMM 701 encoding α-PsGal was transformed in the *Escherichia coli* strain Rosetta (DE3). Heterological expression was carried out at optimal conditions as described previously [36]. Purification of the recombinant α-PaGal was performed according to the procedures described in the reference [35].

#### 3.5.2. Production and Purification of Recombinant α-Nacetylgalactosaminidase

The recombinant wild-type α-Nacetylgalactosaminidase α-NaGa was produced as described earlier [35]. The plasmid DNA pET-40b(+) containing insertion of the gene from the marine bacterium *Arenibacter latericius* KMM 426<sup>T</sup> encoding α-NaGa was transformed in the *Escherichia coli* strain Rosetta (DE3). Heterological expression was carried out at optimal conditions as described previously [37].

The new purification procedure was modified and carried out at 4 ◦C. The cleared supernatant containing α-NaGa and 20% glycerol was loaded directly onto a Ni-sepharose column (5 cm × 36 cm) equilibrated with the buffer A (10 mM NaH2PO4, 10 mM Na2HPO4, 0.5 M NaCl, 5 mM imidazole, 20% glycerol, pH 8.0). The recombinant protein was eluted with the 5–500-mM linear imidazole gradient. The eluted fractions were analyzed, collected and dialyzed against the buffer B (10 mM NaH2PO4, 10 mM Na2HPO4, 50% glycerol, pH 8.0). Then, the protein solution was loaded onto a column (2 cm × 15 cm) with an ion-exchange resin Source 15Q equilibrated with the buffer B. The recombinant protein was eluted with the 0–1.5 M linear NaCl gradient. The fractions exhibiting the activity of α-NaGa were collected and examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

#### 3.5.3. Enzyme and Protein Assays

The activity of α-PsGal and α-NaGa were determined by increasing the amount of p-nithrophenol (pNP). The mixtures containing 50 μL of an enzyme solution and 100 μL of a substrate solution (1 mg/mL) in 0.05 M sodium phosphate buffer (pH 7.0) were incubated at 20 ◦C during 5 min for α-PsGal and 30 min for α-NaGa. The reactions were stopped by the addition of 150 μL of 1 M Na2CO3. One unit of the activity (U) was determined as the amount of an enzyme that releases 1 μmol of pNP per 1 min at 20 ◦C. The amount of the released pNP was determined spectrophotometrically (<sup>ε</sup>400 = 18,300 M−<sup>1</sup> cm<sup>−</sup>1). The specific activity was calculated as U/mg of protein. The protein concentration was determined by the Bradford method calibrated with BSA as a standard [38]. Buffer solutions of α-PsGal (0.1 U/mL) and α-NaGa (0.05 U/mL) were used in the further experiments.

#### *3.6. Effects of Pentacyclic Guanidine Alkaloids on Glycosidases of Marine Bacteria*

#### 3.6.1. The Effects of Monanchomycalin B, Monanchocidin A, and Normonanchocidin A on Glyctosidases

To study the effect of pentacyclic guanidine alkaloids on α-PsGal and α-NaGa, 25 μL of an aqueous solution of monanchomycalin B, monanchocidin A, or normonanchocidin A (1 mg/mL) was mixed with 50 μL of the enzyme solutions in wells of the 96-cell plates and incubated for 30 min. Reactions were initiated by the addition of 75 μL of substrate solutions (p-nithrophenil galactopyranoside (pNP-α-Gal) for α-PsGal and p-nithrophenil N-acetylgalactosaminide (pNP-α-NAcGal) for α-NaGa) in 0.05 M sodium phosphate buffer (pH 7.0). The reaction mixture was incubated at 20 ◦C for 2–30 min in the final volume 150 μL, and then 150 μL of 1M Na2CO3 solution was added to the incubation mixture to stop the reaction. Each reaction mixture was prepared in duplicate. The absorbance was measured at 400 nm. Results were read with a Gen5 and treated with ExCel software. The activity of α-PsGal or α-NaGa was determined as described above. The residual activity was calculated as the ratio v/v0 (%), where v is the enzyme activity in the presence of an inhibitor, and v0 is the enzyme activity in the absence of an inhibitor. The v0 was taken for 100%.

#### 3.6.2. The Irreversibility of Monanchomycalin B Inhibition

To determine the reversibility of the inhibition of the α-PsGal activity, 40 μL (18 μM, H2O) of the monanchomycalin B solution was added to 60 μL of the enzyme solution; the mixture was incubated for 60 min. Two volumes of 20 μL were taken from the reaction mixture, 380 μL of a pNP-α-Gal solution (3.32 mM, in probe was ~5 Km) was added to each mixture, and then the reaction was stopped by addition of 0.6 mL of 1M Na2CO3 after 30 min of incubation. The value of optical

density at the wavelength 400 (OD400) was measured in the 1-cm cuvette. The activity of α-PsGal was determined as described above. The remaining 60 μL of the reaction mixture was dialyzed against 1 L of 0.02 M sodium phosphate buffer (pH 7.0) for 72 h at 4 ◦C. To estimate the dilution, the volume of the reaction mixture after dialysis was measured. The enzyme activity was determined as described above and recalculated taking the dilution into account (1.7 times). A sample of α-PsGal untreated by monanchomycalin B (60 μL of the enzyme solution and 40 μL of H2O) was used as a control. The experiment was carried out in two replicates. The residual activity was calculated as described above.

#### 3.6.3. The Assay of α-PsGal Inhibition by Monanchomycalin B, Monanchocidin A, and Normonanchocidin A

For the α-PsGal inhibition assay, 50 μL of the enzyme solution in 0.05 M sodium phosphate buffer (pH 7.0) were placed in the cells of the 96-well plate with 10 μL of a compound water solution at various concentrations in probes (186, 92.8, 46.4, 23.2, 11.6, 5.8, 2.9, 1.3, 0 μM were for monanchomycalin B; 171.5, 85.7, 42.9, 21.4, 10.7, 5.4, 2.7, 0 μM were for monanchocidin A, and 191.1, 95.6, 47.8, 23.9, 11.9, 6.0, 3.0, 1.5, 0 μM were for normonanchocidin A), and incubated for each concentration during 5, 10, 15, 20, and 25 min. The enzyme reaction was initiated by the addition of 90 μL of the pNPα-Gal solution (3.32 mM, ~5 K m for probe) in 0.05 M sodium phosphate buffer (pH 7.0). The reaction mixtures were incubated for 2 - 15 min in the final volume 150 μL, then 150 μL of the water solution of Na2CO3 (1 M) were added to stop the reaction, and OD400 for the reaction mixtures were immediately measured by a microplate spectrophotometer. The time of each reaction was strictly monitored by stopwatch. The standard and residual activity v/v0 were calculated as described above.

#### 3.6.4. The Kinetic Parameters of Inactivation

The equilibrium inhibition constants ( *K*i) and kinetic inactivation constants (*k*inact) were determined by the classical methods [39]. The inactivation of α-PsGal by the different concentrations of inhibitors (1.3–50 μM) was performed in 0.05 M sodium phosphate buffer (pH 7.0) at a temperature 20 ◦C. An aqueous solution (25 μL) of the compound at the different concentrations was added to 50 μL of the α-PsGal solution (0.2 U/mL), held for 5, 10, 15, 20, and 25 min at 20 ◦C, then 100 μL of the pNPα-Gal solution was added and incubated for 2–15 min at 20 ◦C. The same conditions were used in the control reaction, but the inhibitor was replaced with distilled water. The reactions were stopped by the addition of 1 M Na2CO3 (150 μL); the amount of pNP formed in 1 min was determined as described above. The residual activity v/v0 was presented as a function of time. The pseudo-first-order rate constant of inactivation (*k*obs) was determined for each inactivator concentration as the slope of the v/v0 dependence on the incubation time in semilogarithmic coordinates. The ExCel software was used for these calculations. The second order rate constants for the inactivation process were determined by fitting the dependences of the *k*obs values on the concentration of the inactivators to the Hill's equations. An analysis of the curves and the choice of models for calculation of *K*i (μM) and *k*inact (min–1) were performed with the Origin 8.1 software (OriginLab, Northampton, MA, USA).

#### 3.6.5. Protection of α-PsGal Inactivation by D-galactose

The active-site-directed nature of the inactivation was confirmed by demonstrating protection against the inactivation by competitive inhibitor D-galactose. Inactivation mixtures (75 μL) containing 50 μL of the enzyme solution and 10 μL of D-galactose (0.7 mM in mixture) were preincubated for 15 min, then 15 μL of the monanchomycalin B solution (11.4 μM and 14.2 μM in mixture) were added and incubated at various time intervals as described above. The residual activity of the enzyme was assayed as described above.

#### *3.7. Theoretical Models of α-PsGal Complexes with Guanidine Alkaloids*

The target-template alignment customization of the modeling process and 3D model building of α-PsGalA (GenBank: ABF72189.2) were carried out using the Molecular Operating Environment version 2018.01 [37] package using the forcefield Amber12: EHT. The α-D-galactosidase from *Lactobacillus acidophilus* NCFM (Protein data bank (PDB) code: 2XN2) with a high-resolution crystal structure was used as a template. The evaluation of structural parameters, contact structure analysis, physical-chemical properties, molecular docking, and visualization of the results were carried out with the Ligand interaction and Dock modules in the MOE 2018.01 program. The results were obtained using the equipment of Shared Resource Center Far Eastern Computing Resource of Institute of Automation and Control Processes Far Eastern Branch of the Russian Academy of Sciences (IACP FEB RAS) [40].
