Sulfated steroids of Halichondriidae family sponges – Natural inhibitors of polysaccharide-degrading enzymes of bacterium Formosa algae, inhabiting brown alga Fucus evanescens

Carbohydrate Research 484 (2019) 107776

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Sulfated steroids of Halichondriidae family sponges – Natural inhibitors of polysaccharide-degrading enzymes of bacterium Formosa algae, inhabiting brown alga Fucus evanescens

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Alexey A. Belika, , Kseniya M. Tabakmakhera, Artem S. Silchenkoa, Tatiana N. Makarievaa, C.V. Minhb, Svetlana P. Ermakovaa, Tatiana N. Zvyagintsevaa ⁎

a

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok, 690022, Russia Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam

b

ARTICLE INFO

ABSTRACT

Keywords: Sodium alginate Laminaran Fucoidan Halistanol sulfate Topsentiasterol sulfate D Chlorotopsentiasterol sulfate D

Inhibiting effects of sulfated steroids from marine sponges of Halichondriidae family: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D were investigated on three different types of enzymes degrading polysaccharides of brown algae: endo-1,3-β-D-glucanase GFA, fucoidan hydrolase FFA2 and bifunctional alginate lyase ALFA3 from marine bacterium Formosa algae KMM 3553T, inhabiting thalli of brown alga Fucus evanescens. This is the first research, devoted to influence of a marine natural compound on three functionally related enzymes that make up the complex of enzymes, necessary to degrade unique carbohydrate components of brown algae. Alginic acid, 1,3-β-D-glucan (laminaran) and fucoidan jointly constitute practically all carbohydrate biomass of brown algae, so enzymes, able to degrade such polysaccharides, are crucial for digesting brown algae biomass as well as for organisms surviving and proliferating on brown algae thalli. Halistanol sulfate irreversibly inhibited native endo-1,3-β-D-glucanases of marine mollusks, but reversibly competitively inhibited recombinant endo-1,3-β-D-glucanase GFA. This fact indicates that there are significant structural differences between the enzymes of practically the same specificity. For alginate lyase and fucoidan hydrolase halistanol sulfate was irreversible inhibitor. Topsentiasterol sulfate D was less active inhibitor whereas chlorotopsentiasterol sulfate D was the strongest inhibitor of enzymes under the study. Chlorotopsentiasterol sulfate D caused 98% irreversible inhibition of GFA. Chlorotopsentiasterol sulfate D also caused reversible and 100% inhibition of ALFA3, which is unusual for reversible inhibitors. Inhibition of FFA2 was complete and irreversible in all cases.

1. Introduction Brown algae (Phaeophyta) present an important source of carbohydrates for marine animals and bacteria. Among polysaccharides synthesized by these alga, the most significant moiety belong to alginic acids – up to 45% of dry algal biomass [1], laminarans - up to 34% [2,3] and fucoidans – up to 25% [4]. Inhibitors are necessary for investigation of mechanism of action of enzymes and elucidation of the structure of substrates. Consequently, studies of enzymes: 1,3-β-D-glucanases, fucoidanases and alginate lyases, are of rising importance. This includes their inhibition with compounds, biosynthesized by different marine organisms. Alginate lyases are widespread in nature and were found in marine algae and invertebrates, fungi, bacteria and some viruses [5]. Alginate ⁎

lyases digest substrate by the mechanism of β-elimination. According to the classical enzyme nomenclature, alginate lyases are divided to polyguluronate lyases (EC 4.2.2.11) [6,7], polymannuronate lyases (EC 4.2.2.3) [8] and bifunctional alginate lyases [9], which are specific to cleave 1→4-glycosidic bonds between both residues of mannuronic and guluronic acids [10–12]. By the mechanism of action, alginate lyases are divided to exo- and endo-enzymes. The majority of alginate lyases are of endo-type of action, which leads to formation of mixture of oligosaccharides. Any data about specific inhibition of alginate lyases with natural low-molecular compounds has not been found in literature. On the other hand, significant decrease of activity of these enzymes can be caused by ions of silver, bivalent metals, glutathione and, in case of metal-dependent alginate lyases, EDTA and EGTA [13,14]. Fucoidan hydrolases (fucoidanases) catalyze hydrolysis of glycoside

Corresponding author. E-mail address: [email protected] (A.A. Belik).

https://doi.org/10.1016/j.carres.2019.107776 Received 1 July 2019; Received in revised form 5 August 2019; Accepted 9 August 2019 Available online 09 August 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.

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bonds between residues of α-L-fucose in fucoidan molecules. On the base of CAZy classification, fucoidanases belong to GH 107 family of glycoside hydrolases. It's necessary to highlight that fucoidan hydrolases belong to the less studied polysaccharide-degrading enzymes [15]. There is only spare data about inhibitors of fucoidanases in literature. Thus, inhibitors of recombinant fucoidanase of marine bacterium F. algae KMM 3553T were found among metabolites of algae Fucus evanescens and Costaria costata [15]. Interest to fucoidanases and their inhibitors is growing by the reason, that they can be used as instruments in study of complex molecules of fucoidans with wide spectra of biological activities [16–18] and for producing of biologically active fucooligosaccharides, as they are more convenient for organisms than high-molecular fucoidans. Endo-1,3-β-D-glucanases (EC 3.2.1.39) present wide group of enzymes with great importance for assimilation of carbohydrate nutrients, cell growth and differentiation, protection from pathogenic organisms. These enzymes are distributed among seven structural families of Oglycoside hydrolases. Data about organic or metal-organic inhibitors of such enzymes is very limited [19–21]. So, it is know, that endo-1,3-β-Dglucanases from bivalvia are specifically inhibited by sulfated polyoxysteroids from marine sponges, starfishes and ophiuras. It is shown that the effectiveness of inhibition depends on structural peculiarities of the substances [22–25]. The most study was done for inhibition of endo-1,3-β-D-glucanases from bivalvia Pseudocardium (Spisula) sachalinensis and Chlamys albidus. Strong inhibiting influence of halistanol sulfate on endo-1,3-β-D-glucanases from Ch. albidus, P. sachalinensis and P. yessoensis was detected even in 1 μM concentration [26]. There was shown, that chemical modifications of lysine and histidine in endo-1,3β-D-glucanase from P. sachalinensis protect this enzyme from inactivating action of halistanol sulfate, that is evidence of probable role of these amino acid residues in binding with the inhibitor. The studies of spectra of differential UV absorption of endo-1,3-β-D-glucanase from P. sachalinensis revealed their similarity with ones, appearing after binding of enzyme with laminaran, that is evidence of perturbation of amino acid residues in enzyme's active center caused by halistanol sulfate. The results of intrinsic fluorescence, UV absorption spectrophotometry, and CD spectroscopy showed that halistanol sulfate causes conformational changes in tertiary structure of endo-1,3-β-D-glucanases from bivalvia, satelliting with exposure of tyrosine residues in side chains and partial immersing of surface residues of tryptophan. Halistanol sulfate (1) (Fig. 1) is known as a compound with irreversible inhibiting action on endo-1,3-β-D-glucanases of marine mollusks [25]. Closely related (by chemical structure) trisulfated polyoxysteroids topsentiasterol sulfate D (2) and chlorotopsentiasterol sulfate D (3) (Fig. 1) showed, like (1), ability to inhibit endo-1,3-β-Dglucanase from marine mollusk P. sachalinensis and did not show significant effect on endo-1,3-β-D-glucanase from fungus Chaetomium indicum [27]. Studied inhibitors 1–3 belong to the same class of natural substances - 2β,3α,6β-trisulfated polyoxysteroids. Today, this class consists of more than 40 representatives, isolated from tropical marine sponges, predominately from Halichondriidae family [28]. Common structural peculiarity of compounds 1–3 and their relatives is their amphiphilicity. They have strongly polarized hydrophilic part, consisting of three sulfate anions and very weakly polarized hydrophobic part, consisting of steroid nucleus and side chain. As amphiphilic compounds, they have slight structural similarity with such detergents, such as sodium dodecyl sulfate with one sulfate group and weakly polarized alkyl hydrophobic part. Halistanol sulfate and other natural trisulfated steroids are known for a wide range of inhibiting activity. For example, there was shown inhibitory activity against NAD+-dependent lysine deacetylase (KDAC) – sirtuins (SIRT) [29,30]. So, it was a reasonable idea to test the inhibitory activity of trisulfated polyoxysteroids on carbohydrate-degrading enzymes. There have not been previous studies of these or other trisulfated polyoxysteroids on microbial natural or recombinant endo1,3-β-D-glucanases, fucoidan hydrolases, alginate lyases.

Fig. 1. Structural formulas of halistanol sulfate (1), topsentiasterol sulfate D (2) and chlorotopsentiasterol sulfate D (3).

Because trisulfated steroids have hydrophilic sulfate groups and hydrophobic aliphatic side chains and can be considered as surface active compounds. So, it's necessary to make control experiments with other surfactants (SDS, for example) to determine the real background of the inhibition. It can be understood by the way, that real inhibitors deactivate the enzymes in much less concentrations than surfactants, because they interact with enzyme's active center and not just denaturize its 3D structure, like SDS. This article is dedicated to the study of action of sulfated steroids isolated from Halichondriidae family sponges: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D on recombinant endo-1,3-β-D-glucanase, fucoidan hydrolase and bifunctional alginate lyase of marine bacterium F. algae KMM 3553T – a complex of microbial enzymes, taking parts in degradation of brown seaweed polysaccharides. This is the first research of several structurally related compounds, where each of them is an effective inhibitor of each enzyme from bacterial polysaccharide-degrading complex, necessary and sufficient to utilize all carbohydrates of brown algae. 2. Results There should be used different methods for detection of enzymatic activity: reducing sugars assaying (Nelson's method) for endo-1,3-β-Dglucanase GFA, 235 nm optical density assaying for alginate lyase ALAF3 and C-PAGE for fucoidan hydrolase FFA2. This is because glucose and laninariologosaccharides are the main products of GFA, alginate oligosaccharides with fluorescent 4-deoxy-L-erytro-hex-4-enopyranosiluronate link are the products of ALFA3, and fucooligosaccharides of different degree of polymerization are the 2

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different from their action on GFA. Halistanol sulfate provided complete and irreversible inhibition. Dilution of mixture (E + S + I) in 8 times did not change specific activity of ALFA3, while it caused reversible action on GFA. Topsentiasterol sulfate D provided partial inhibition. Chlorotopsentiasterol sulfate D completely inhibited ALFA3 at concentration of inhibitor 0.46 mM. Both compounds demonstrated reversible type of inhibition: specific activity of alginate lyase increased in 2.4 times in case of topsentiasterol sulfate D (2) and in 2 times in case of chlorotopsentiasterol sulfate D (3). It's possible to highlight complete inhibition of ALFA3 by chlorotopsentiasterol sulfate D (3) that is unusual for reversible inhibitor. Usually, reversible inhibitors do not cause complete inhibition of enzymes. Sodium dodecyl sulfate did not cause any significant inhibition of recombinant GFA and ALFA3 even in concentrations up to 0.29% (w/ v). Thus, inhibiting action of sulfated steroids is not connected with surface active properties of these molecules. Action of compounds 1–3 on catalytic activity of recombinant fucoidanase FFA2 was investigated with polyacrylamide gel electrophoresis (C-PAGE) as method of detection of fucoidanase activity. It was shown, that these compounds inhibit activity of FFA2 at different degree (Table 2, Figs. 2 and 3). Thus, values of IC50 for compounds (1), (2) and (3) were 0.165, 0.41, and 0.185 mM, respectively (Fig. 3). Dilution of mixture of enzyme and inhibitor led to slight decrease of activity of enzyme in compare with control (without addition of compounds 1–3) that indicates possible irreversible mechanism of inhibition of fucoidanase FFA2. The obtained data on inhibition of different microbial recombinant enzymes with compounds 1–3 in compare with results of treating of marine mollusks’ endo-1,3-β-D-glucanases with the same compounds are summarized in Table 3.

Table 1 Dependence of degree of inhibition of endo-1,3-β-D-glucanase GFA from concentration of sulfated steroids. Enzyme GFA, [C], μM Substrate, [C], mg/ml

0.4 0.8

Halistanol sulfate (1) Inhibitor, [C], mM Specific activity, U/mg Degree of inhibition, %

0 0.47 0

0.03 0.46 2

0.13 0.45 5

0.26 0.41 13

0.53 0.38 20

1.06 0.28 42

2.04 0.24 51

3.80 0.24 59

0.01 0.49 0

0.02 0.49 1

0.03 0.47 4

0.06 0.45 8

0.13 0.41 16

0.25 0.35 29

0.51 0.31 38

0.02 0.47 0

0.03 0.48 0

0.06 0.40 16

0.12 0.38 20

0.24 0.14 70

0.48 0.01 98

Topsentiasterol sulfate D (2) Inhibitor, [C], mM Specific activity, U/mg Degree of inhibition, %

0 0.49 0

Chlorotopsentiasterol sulfate D (3) Inhibitor, [C], mM Specific activity, U/mg Degree of inhibition, %

0 0.46 0

0.01 0.4 0

products of FFA2. It's not possible to assay the changes of activity of FFA2 by spectrophotometry, but the change of products composition, seen on C-PAGE, is quite evident. The type of inhibition was determined with dilution of preincubated mixture of enzyme and inhibitor, because of the fact that reversibly inhibited enzyme loses weakly bound molecules of inhibitor in diluted mixture and the enzyme partly restores its activity, and doesn't in the case of irreversible inhibition. All inhibitors were completely soluble in mentioned buffers in concentrations up to 10 mg/ml. The inhibitors did not cause any foam formation in mentioned concentrations. Their influence on the optical densities of reaction mixtures was neutralized by adding them to controls in the same concentrations. Investigation of inhibition of endo-1,3-β-D-glucanase GFA by halistanol sulfate showed that maximal inhibition of enzyme's activity did not exceed 59% (Table 1). Dilution of reaction mixture let to increase of enzyme's specific activity that indicates reversible binding of inhibitor with enzyme. Investigation of dependence between concentration of substrate and speed of the reaction, done by Lineweaver-Burk method, indicated that inhibition is competitive. Constant of inhibition (Ki) was calculated using Dixon plot and appeared to be 0.6 mM. Thus, halistanol sulfate is reversible competitive inhibitor of microbial endo-1,3-βD-glucanase GFA, though it's irreversible inhibitor for endo-1,3-β-Dglucanases of marine mollusks [25]. Background of such differences is probably in significant structural differences of microbial and mollusks' endo-1,3-β-D-glucanases. Nevertheless, it's possible to suppose that, despite the scale, these differences do not influence on substrate specificities of these enzymes. Investigation of interaction of endo-1,3-β-D-glucanase GFA with topsentiasterol sulfate D revealed that topsentiasterol sulfate D is weak inhibitor, causing maximal inhibition of GFA no more than 38%. Topsentiasterol sulfate D differs from halistanol sulfate by presence of additional hydroxyl group in steroid nucleus and furan five-member cycle in side chain. It is possible that furan residue makes the inhibitor less hydrophobic that weakens inhibitor's binding with hydrophobic amino acid residues of the enzyme and decreases its inhibiting action. Presence of chlorine in chlorotopsentiasterol sulfate D (Fig. 1) increases hydrophobicity of side chain and causes practically complete inhibition of GFA (Table 1). Furthermore, presence of chlorine in the molecule of sulfated steroid changes the type of inhibition from reversible to irreversible in compare with halistanol sulfate (specific activity of GFA did not increase along with dilution of mixture “enzyme + chlorotopsentiasterol sulfate D″). Inhibition of alginate lyase ALFA3 with sulfated steroids appeared to be acted by different mechanisms and strongly depended on inhibitor structure, similarly to GFA. But their action on alginate lyase was

3. Discussion There was investigated action of halistanol sulfate (1) and its analogues - topsentiasterol sulfate D (2) and chlorotopsentiasterol sulfate D (3) (Fig. 1) on three types of enzymes degrading different substrates: endo-1,3-β-D-glucanase GFA – laminaran from brown alga S. cichorioides, alginate lyase ALFA3 - sodium alginate, fucoidanase FFA2 - 1 → 3; 1→4-α-L-fucan from brown alga F. evanescens. These enzymes were selected by the reason that their substrates comprise the whole complex of water-soluble polysaccharides of brown algae and, thus, the enzymes are involved in algal biomass consumption by microorganisms. Their importance in algal biomass degradation can be illustrated by the fact that alginic acids can comprise up to 45% dry algal biomass [1], laminarans - up to 34% [2,3] and fucoidans – up to 25% [4]. So, altogether these three enzymes can utilize up to 90% of dry brown algal biomass. The industrial applications of these enzymes vary from bioethanol production to obtaining oligosaccharides with high therapeutic potential [31–33]. The substrates were selected because they appeared to be the most suitable and available for these enzymes of all tested substrates, as it Table 2 Dependence of degree of inhibition of fucoidanase FFA2 from concentration of sulfated steroids. Enzyme FFA2, [C], μM Substrate, [C], mg/ml Halistanol sulfate (1), [C], mM Degree of inhibition, % Topsentiasterol sulfate D (2), [C], mM Degree of inhibition, % Chlorotopsentiasterol sulfate D (3), [C], mM Degree of inhibition, %

3

1.44 10 0.00

0.07

0.13

0.27

0.53

0.80

1.06

1.33

0 0.00

8 0.06

37 0.13

77 0.25

89 0.51

96 0.76

100 1.01

100 1.26

0 0.00

6 0.06

10 0.12

18 0.24

72 0.48

80 0.73

84 0.97

100 1.21

0

8

11

72

91

92

100

100

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Fig. 2. Electropherogram of the products of hydrolysis of fucoidan from F. evanescens catalyzed by FFA2 in the presence of compounds (1), (2), and (3). Numbers at the top of gels refer to the micromolar concentration of compounds 1–3. Specific activity of FFA2 was calculated from integrated optical density of enzymatic hydrolysis products (ladders of bands on electropherograms) quantified by using Quantity One software. Calculated values of residual activity are indicated below the gels. Ci – control of inhibitor. The optical density of reaction products without the addition of any compounds to the reaction mixture was chosen as 100%. Residual activities were calculated separately for each dilution.

was shown in previous studies. In brief, GFA exhibited the highest activity on S. cichorioides laminaran [34], ALFA3 digested commercial Sigma-Aldrich sodium alginate more rapidly and in wider range of conditions than any other alginate (unpublished data), and it was also shown that FFA2 catalyzed cleavage of fucoidan from F. evanescens to form low molecular weight products (LMP) and a polymeric fraction (HMP) with 50.8 kDa molecular weight and more than 50% yield [35]. Inhibitors of these enzymes can be potential regulators of such consumption and protect algae from destructing action of microorganisms.

Previously it was shown, that inhibiting action of halistanol sulfate (1) on endo-1,3-β-D-glucanases of marine mollusks is severe and irreversible [25]. Modifications of hydrophilic and hydrophobic parts of halistanol sulfate weakened inhibition [25,27,36], but the type of inhibition remained the same. The fact that halistanol sulfate (1) is reversible and competitive inhibitor of bacterial endo-1,3-β-D-glucanase GFA was unexpected. This indicates significant differences in 3D structures of glucanases from mollusks and bacteria, though they have comparable molecular masses and their specificities remain practically

Fig. 3. Concentration-dependent inhibition curves of FFA2 in presence of compounds 1–3. Residual activities of FFA2 in presence of compounds 1–3 were calculated from integrated optical density of enzymatic hydrolysis products and quantified by using Quantity One software. The enzyme activity of FFA2 without addition of compounds 1–3 corresponds to 100% in the plot. Calculated IC50 values 165, 410, and 185 μM correspond to concentrations of compounds 1–3, respectively. 4

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4. Material and methods

Table 3 IC50, type and maximal degree of inhibition of enzymes from marine mollusks and bacterium F. algae by sulfated steroids. Standard deviations are given. Source of enzyme

Marine mollusks (Ch. albidus, P. yessoensis, P. sachalinensis)

Marine bacterium F. algae, recombinant enzymes

Enzyme

endo-1,3-β-Dglucanases [25]

endo-1,3-βD-glucanase (GFA)

fucoidanase (FFA2)

alginate lyase (ALFA3)

Irreversible

Reversible, competitive 59% 0.203

Irreversible

Irreversible

100% 0.165

100% 0.13

Irreversible 38% –

Irreversible 100% 0.41

Reversible 58% 0.46

Irreversible 98% 0.180

Irreversible 100% 0.185

Reversible 100% 0.12

4.1. Reagents Halistanol sulfate (1) was isolated from sponge Halichondria sp., collected near the shores of Madagascar Island during the 12th expedition on research vessel “Professor Bogorov” [36]. Topsentiasterol sulfate (2) and chlorotopsentiasterol sulfate (3) were isolated from marine sponge Topsentia (Halichondria) sp., collected in Van Phong bay during the 49th expedition on research vessel “Akademik Oparin” and identified by NMR and other methods [27,37]. Structural formulae of these compounds are presented on Fig. 1. Recombinant endo-1,3-β-Dglucanase of marine bacterium F. algae (GFA) was produced by expression in Escherichia coli by the previously described method [34]. Laminaran from brown algae Saccharina cichorioides was produced by previously described method [38]. Recombinant fucoidan hydrolase (FFA2) and alginate lyase of marine bacterium F. algae (ALFA3) produced by expression in E. coli by the previously described methods [34]. Fucoidan from brown algae F. evanescens was produced by previously described method [39]. Sodium alginate (M/G estimately 3:1) and other reagents were commercial supplies from Sigma-Aldrich (USA) and Helicon (Russia).

Halistanol sulfate (1) Type of inhibition Maximal degree IC50, mM Topsentiasterol sulfate D (2) Type of inhibition Maximal degree IC50, mM

Irreversible

Chlorotopsentiasterol sulfate D (3) Type of inhibition Maximal degree IC50, mM

Irreversible

4.2. Instrumental Optical densities was measured on BioTek PowerWave™ XS spectrophotometer (USA), 96-well plates were used. C-PAGE was done in GE Healthcare SE 600 Ruby electrophoresis chamber (USA) and GE Healthcare EPS-301 power supply (USA). Gel images were visualized using a calibrated densitometer Bio-Rad DS-800 (USA). ТS-1/80-SPU (Russia) and TT-2 Termit (Russia) thermostats were used during incubation of reaction mixtures.

the same. Topsentiasterol sulfate D (2), that has the least hydrophobic side chain among the studied compounds is the weakest inhibitor of endo-1,3-β-D-glucanase (GFA), fucoidanase (FFA2) and alginate lyase (ALFA3). Inhibition of ALFA3 with chlorotopsentiasterol sulfate D (3) is relatively rare case of complete, but reversible inhibition and is worth further investigation. Data of molecular docking support the affinity of sulfated steroids to active centers of all mentioned enzymes. Analysis of the results of molecular docking showed that full fitness of binding of all sulfated steroids with active centers of GFA and ALFA3 was in range from −1500 to −1700 kcal/mol, change of free Gibbs energy was in range from −7 to −10 kcal/mol without significant differences from particular compound or enzyme. Full fitness of binding of all sulfated steroids with active center of FFA2 was about 3200 kcal/mol, change of free Gibbs energy was from −8 to −10 kcal/mol, which can be a probable reason why their inhibition of FFA2 was complete and irreversible. So, binding if inhibitor with active center of enzyme was one of the most preferable (in compare with binding with other sites of the surface), but not outstandingly preferable. This is the probable reason, why inhibition is achieved in relatively great concentrations of compounds 1–3 about 1000 molecules of inhibitor to 1 molecule of enzyme. As conclusion, the greatest importance in strength and type of inhibition by sulfated steroids can be attributed to hydrophobicity and size of aliphatic their side chains. For example, compound (3) differs from (2) only with chlorine atom in the side chain, but shows much greater inhibiting activity. With the greatest probability, this inhibition is caused with binding of steroid's side chain with enzyme's active center [30] and is not related with surface active properties of molecules of sulfated steroids, as SDS did not significantly influence on the enzyme's activity even in concentrations 10 times greater than it was necessary for sulfated steroids to achieve complete inhibition. Using the collection of sulfated steroids demonstrated not only significant differences in structures and mechanisms of action of enzymes with different specificities, but also differences between structures of enzymes with the same specificity, isolated from different organisms - endo-1,3-β-D-glucanases from marine mollusks and bacteria.

4.3. Inhibition of recombinant endo-1,3-β-D-glucanase of marine bacterium F. algae KMM 3553T 4.3.1. Determination of activity of endo-1,3-β-D-glucanase GFA Reaction mixture, containing 20 μl of enzyme solution (0.2 mg/ml) in 0.025 M succinate buffer (pH 5.5) and 100 μl of solution of laminaran from S. cichorioides (1 mg/ml) in the same buffer was incubated at 37 °C during 30 min. Reaction was stopped by heating up to 80 °C during 5 min. Activity of GFA was determined by assaying the amount of appeared reducing sugars by Nelson method [40]. Optical density was measured at λ = 750 nm. Concentration of reducing sugars was determined by calibration curve, using glucose as standard. 4.3.2. Inhibition of endo-1,3-β-D-glucanase GFA by compounds 1-3 Solution of endo-1,3-β-D-glucanase was mixed with solutions of compounds 1–3 (10 μl, final concentrations are given in Table 1. Solutions were incubated during 1 h at 24 °C, and then the solution of laminaran from S. cichorioides, the mixture was incubated during 30 min at 37 °C. Aliquot 100 μl was taken and analyzed by Nelson method. 4.3.3. Determination of type of inhibition of endo-1,3-β-D-glucanase GFA Solution of endo-1,3-β-D-glucanase was mixed with solutions of compounds 1–3 (10 μl). Solutions were incubated during 1 h at 24 °C, then aliquots of 20 μl were diluted in 1, 2 and 4 times with succinate buffer (pH 5.5), and mixed with laminaran solution in the same buffer to final concentration of laminaran 1 mg/ml. Reaction mixtures were incubated during 20 min (for initial concentration), 40 min (for 2 times dilution) and 80 min (for 4 times dilution) at 37 °C. Aliquots 100 μl were analyzed by Nelson method. 5

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4.4. Inhibition of recombinant alginate lyase of marine bacterium F. algae KMM 3553T

with server Swiss-Model and using of PDB 4bow.1.A, 5zu5.1.A, 6dlh.1 as templates of 3D structures, respectively [42]. Models quality was estimated with program pack PROCHECK [43]. Molecular docking of compounds 1–3 and computation of energy of binding were done with server SwissDock [44].

4.4.1. Determination of activity of alginate lyase ALFA3 Solution of recombinant bifunctional alginate lyase ALFA3 was mixed with solution of sodium alginate, reaction was done at 37 °C during different time intervals. Formation of double bond between C4–C5 atoms was registered with spectrophotometer at 235 nm wavelength [31].

Acknowledgements The work on investigation of structures and mechanisms of action of enzymes was supported by RFBR grant № 18-04-00905_а. The part of this work on isolation and structural elucidation of sulfated steroids of Halichondriidae family sponges was supported by the RFBR grant №1853-54002 Viet-a.

4.4.2. Inhibition of alginate lyase ALFA3 Solution of alginate lyase ALFA3 was taken 0.33 μM, solutions of inhibitors were prepared to concentrations 0.002–0.49 mg/ml, and then the aliquots of 20 μl of enzyme and 10 μl of inhibitor were mixed and incubated at 24 °C during 1 h. After adding solution of sodium alginate reaction was done at 37 °C during different time intervals. Reaction products were registered with spectrophotometer at 235 nm wavelength (see above).

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4.4.3. Determination of type of inhibition of alginate lyase ALFA3 Aliquots of solution of ALFA3 were mixed with solutions of compounds 1–3 in concentrations 0.58, 0.486 and 0.233 mg/ml, respectively (10 μl, in the same buffer). Mixtures were incubated during 1 h at 24 °C and then aliquots of 20 μl were diluted in 1, 2 and 4 times with succinate buffer (pH 6.0). Prepared samples were incubated at 24 °C during 1 h, then solution of sodium alginate in the same buffer was added up to final concentration 4 mg/ml. Reaction mixtures were incubated during 20 min (for initial concentration), 40 min (for 2 times dilution) and 80 min (for 4 times dilution) at 37 °C. Aliquots were analyzed with spectrophotometer at 235 nm wavelength (see above). 4.5. Inhibition of recombinant fucoidanase of marine bacterium F. algae KMM 3553T 4.5.1. Determination of activity of fucoidanase FFA2 Activity of fucoidanase FFA2 was determined by electrophoresis in a polyacrylamide gel (C-PAGE) according to the literature [41]. Mixture of enzyme and fucoidan solutions was incubated at 34 °C for 30 min. The reaction was stopped by heating at 80 °C for 5 min. The hydrolysis products were mixed with loading buffer and separated on C-PAGE. Fucoidanase activity was detected by the occurrence of charged oligosaccharide bands in the gel. 4.5.2. Inhibition of fucoidanase FFA2 A solution of fucoidanase FFA2 was treated with an compounds 1–3 solution at concentrations of 0.5–10 mg/ml, incubated for 20 min at room temperature (24 °C), treated with a solution of fucoidan from F. evanescens (20 mg/ml) in the same buffer, and incubated for 30 min at 34 °C. An aliquot (5 μl) of the resulting mixtures were analyzed by CPAGE (see above). 4.5.3. Determination of the type of inhibition of fucoidanase FFA2 A solution of fucoidanase FFA2 was treated with compounds 1–3. Aliquots (10 μl) from resulting mixtures were taken and then diluted by 1, 2, and 4 times with 0.04 M tris-HCl buffer pH 7.0. Resulted samples were incubated for 20 min at room temperature (24 °C) and then treated with a solution of fucoidan from F. evanescens (10 mg/ml, final concentration) in the same buffer. Reaction mixtures were incubated for 1 h (for dilution 1), 2 h (for dilution 2) and 4 h (for dilution 4) at 34 °C. An aliquot (5 μl) of the resulting mixtures were analyzed by polyacrylamide gel electrophoresis (see above). 4.6. Molecular docking 3D models of endo-1,3-β-D-glucanase GFA, alginate lyase ALFA3 and fucoidanase FFA2 were built by method of homology modeling 6

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