Fucanases of the 107’th structural family of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T

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Abstract

This work is devoted to a generalization and comparison of the structural organization, physicochemical properties and substrate specificity of four fucanases of glycoside hydrolase family 107 that are part of the fucoidanutilizing cluster of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The established differences in the significant characteristics of these enzymes provide new knowledge about the characteristics of fucanases belonging to the GH107 family.

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About the authors

Anastasiya O. Zueva

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS

Author for correspondence.
Email: a.o.zueva@yandex.ru
ORCID iD: 0000-0001-9570-7647

Junior Researcher

Russian Federation, Vladivostok

Artem S. Silchenko

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS

Email: artem.silchencko@yandex.ru
ORCID iD: 0000-0002-3502-5692

Candidate of Sciences in Chemistry, Senior Researcher

Russian Federation, Vladivostok

Svetlana P. Ermakova

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS

Email: swetlana_e@mail.ru
ORCID iD: 0000-0002-5905-2046

Doctor of Sciences in Chemistry, Assistant Professor, Head of the Laboratory

Russian Federation, Vladivostok

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Supplementary files

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2. Fig. 1. Schematic representation of the fucoidandegradating gene cluster of the marine bacterium W. fucanilytica CZ1127T

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3. 2. Domain organization of the FWf1‒FWf4 polypeptide chains and characterized fucanases of the GH107 family. The lengths of the amino acid sequences of fucanases are indicated to the right of the schematic representation of their domain organization. For FWf1 and FWf3, they are given both for situations with missing signal sequences (GenBank: ANW96097.1 (FWf1) and ANW96115.1 (FWf3)) and for those (*) in which the latter are present (GenBank: WP_083194609.1 and WP_083194615.1)

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4. 3. Three‒dimensional structures of full-size fucanases FWf1-FWf4 and the previously characterized fucanase FcnA [18], predicted using the AlphaFold service. The domains present in the polypeptide chains of fucanases are indicated in different colors

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5. 4. The region of multiple alignment of the catalytic domains of fucanases FWf1‒FWf4 and known proteins of the GH107 family. The residues of the catalytic amino acids Asp and His are marked with red borders. The signatures of the amino acids RxxxxxxDxxxxD and DxxxGH, conserved for all known representatives of the GH107 family, are indicated at the top of the sequences.

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6. 5. Structural alignment of three-dimensional models of the catalytic domains GH107 of fucanases FWf1‒FWf4 and fucanase FcnA (code PDB: 6DLH). The amino acid residues of the active centers FWf1‒FWf4 and FcnA, which are conserved, are emphasized. The Asp and His residues, which perform a catalytic function, are highlighted in red

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7. 6. Electrophorogram of fucoidan hydrolysis products from F. evanescens obtained using fucanases FWf1‒FWf4 over different incubation times. Cs – fucoidan from F. evanescens, which has not undergone hydrolysis

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8. 7. Analysis of the activity of fucanases FWf1‒FWf4 in relation to fucoidans isolated from various species of brown algae: a diagram of the main structural motifs present in fucoidans isolated from F. evanescens (FeF), U. pinnatifida (UpF), S. cichorioides (ScF), F. vesiculosus (FvF) and S. horneri (ShF); b is a table showing the activity level of FWf1‒FWf4 in relation to FeF, UpF, ScF, FvF or ShF

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9. 8. Electrophorogram (a) and schematic representation of the structures of isolated oligosaccharides, products of enzymatic hydrolysis of fucoidan FeF, obtained using fucanases FWf1 (b), FWf2 (c), FWf3 (d) and FWf4 (e)

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10. 9. Schematic representation of fucoidan FeF depolymerization using FWf1‒FWf4. The red arrow points to sites in FeF that FWf1‒FWf4 fucanases recognize and cleave

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11. 10. Analysis of the activity of fucanases FWf1‒FWf4 in relation to fucooligosaccharides. Electrophorogram of hydrolysis products obtained by treatment with fucanases of various fucooligosaccharides: a – FWf1 and FWf2, b – FWf3 and FWf4; c – table showing the activity level of FWf1‒FWf4 in relation to sulfated oligosaccharides: (−) ‒ fucanase activity was not detected; (+) ‒ low activity; (++) ‒ average activity; (+++) ‒ high activity. The data were obtained from electrophoregrams (a and b). The degrees of polymerization are calculated: (SP = 2*) ‒ based on the proposed depolymerization products of tetrasaccharide 4F2,3,4S(7S) fucanase FWf1; (SP = 6*) ‒ octasaccharide 8F2,3S(12S) fucanase FWf2. Cs – fucooligosaccharides that have not undergone enzymatic hydrolysis

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12. 11. Putative fucoidan cleavage sites recognized by FWf1‒FWf4 endofucanases and fucoidan structural motifs resistant to cleavage by FWf1‒FWf4 fucanases (a). The topology of the putative carbohydrate‒binding subsites of FWf1-FWf4 fucanases (b). The numbers above the structural motifs indicate carbohydrate-binding subsites FWf1‒FWf4. The sub-sites are numbered according to the nomenclature [42]. The asterisk indicates the cleavage site assumed based on the structures of oligosaccharides obtained by hydrolysis of fucoidan FeF using FWf1, FWf3 or FWf4

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