title: Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii.
abstract: The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of β-D-mannuronic acid (M) to α-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.
authors.affiliation: From the Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Frederik Bajers vej 7H, DK-9220 Aalborg, Denmark, the Department of Biotechnology, Norwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
authors.forename: Edith
authors.initials: E
authors.lastname: Buchinger
authors.affiliation: From the Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Frederik Bajers vej 7H, DK-9220 Aalborg, Denmark.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Knudsen
authors.affiliation: the Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000, Denmark, and.
authors.forename: Manja A
authors.initials: MA
authors.lastname: Behrens
authors.affiliation: the Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000, Denmark, and.
authors.forename: Jan Skov
authors.initials: JS
authors.lastname: Pedersen
authors.affiliation: the Department of Biotechnology, Norwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
authors.forename: Olav A
authors.initials: OA
authors.lastname: Aarstad
authors.affiliation: the Department of Bioprocess Technology, SINTEF Materials and Chemistry, N-7465 Trondheim, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Tøndervik
authors.affiliation: the Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000, Denmark, and.
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
authors.affiliation: the Department of Biotechnology, Norwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
authors.forename: Gudmund
authors.initials: G
authors.lastname: Skjåk-Bræk
authors.affiliation: From the Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Frederik Bajers vej 7H, DK-9220 Aalborg, Denmark.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wimmer
authors.affiliation: the Department of Biotechnology, Norwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway, finn.l.aachmann@ntnu.no.
authors.forename: Finn L
authors.initials: FL
authors.lastname: Aachmann
mesh_terms: D000464:Alginates / Q000737:chemistry*; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002151:Calorimetry; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D014965:X-Rays
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D002238:Carbohydrate Epimerases
keywords: Alginate; Alginate C-5 Epimerase; Calcium-binding Protein; Carbohydrate-binding Protein; ITC; Nuclear Magnetic Resonance (NMR); Protein Chemistry; Protein Engineering; R-module; SAXS