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J Biol Chem. 2014 Nov 7;289(45):31382-96. doi: 10.1074/jbc.M114.567008. Epub 2014 Sep 29.

Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii.

Author information

1
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.
2
From the Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Frederik Bajers vej 7H, DK-9220 Aalborg, Denmark.
3
the Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000, Denmark, and.
4
the Department of Biotechnology, Norwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
5
the Department of Bioprocess Technology, SINTEF Materials and Chemistry, N-7465 Trondheim, Norway.
6
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.

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.

KEYWORDS:

Alginate; Alginate C-5 Epimerase; Calcium-binding Protein; Carbohydrate-binding Protein; ITC; Nuclear Magnetic Resonance (NMR); Protein Chemistry; Protein Engineering; R-module; SAXS

PMID:
25266718
PMCID:
PMC4223338
DOI:
10.1074/jbc.M114.567008
[Indexed for MEDLINE]
Free PMC Article

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