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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.

Category

Academic article

Client

  • Research Council of Norway (RCN) / 221576

Language

English

Author(s)

  • Edith Buchinger
  • Daniel H. Knudsen
  • Manja A. Behrens
  • Jan Skov Pedersen
  • Olav Andreas Aarstad
  • Anne Tøndervik
  • Svein Valla
  • Gudmund Skjåk-Bræk
  • Reinhard Wimmer
  • Finn Lillelund Aachmann

Affiliation

  • Norwegian University of Science and Technology
  • Aalborg University
  • Aarhus University
  • SINTEF Industry / Biotechnology and Nanomedicine

Year

2014

Published in

Journal of Biological Chemistry

ISSN

0021-9258

Volume

289

Issue

45

Page(s)

31382 - 31396

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