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J. Biol. Chem., Vol. 281, Issue 11, 7350-7356, March 17, 2006

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NMR Structure of the R-module

A PARALLEL -ROLL SUBUNIT FROM AN AZOTOBACTER VINELANDII MANNURONAN C-5 EPIMERASE^*

Finn L. Aachmann, Britt I. G. Svanem, Peter Güntert^¶, Steffen B. Petersen^||, Svein Valla¹, and Reinhard Wimmer²

From the Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark, Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway, ^¶Tatsuo Miyazawa Memorial Program, RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan, and ^||Institute of Physics and Nanotechnology, Aalborg University, Skjernvej 4A, DK-9000 Aalborg, Denmark

In the bacterium Azotobacter vinelandii, a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7) has been identified. These epimerases are responsible for the epimerization of -D-mannuronic acid to -L-guluronic acid in alginate polymers. The epimerases consist of two types of structural modules, designated A (one or two copies) and R (one to seven copies). The structure of the catalytically active A-module from the smallest epimerase AlgE4 (consisting of AR) has been solved recently. This paper describes the NMR structure of the R-module from AlgE4 and its titration with a substrate analogue and paramagnetic thulium ions. The R-module folds into a right-handed parallel -roll. The overall shape of the R-module is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicated possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule and the importance of calcium binding for the stability of the R-module. Structure calculations showed that calcium ions can be incorporated in these loops without structural violations and changes. Based on the structure and the electrostatic surface potential of both the A- and R-module from AlgE4, a model for the appearance of the whole protein is proposed.

Received for publication, September 13, 2005 , and in revised form, December 13, 2005.

The atomic coordinates and structure factors (code 2AGM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was financed by the Danish Technical Research Council (STVF) and the Norwegian Research Council (NFR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹To whom correspondence may be addressed: Dept. of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. Tel.: 47-73-59-86-94; Fax: 47-73-59-12-83; E-mail: svein.valla{at}biotech.ntnu.no. ²To whom correspondence may be addressed: Dept. of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. Tel.: 45-96-35-85-18; Fax: 45-98-14-18-08; E-mail: rw{at}bio.aau.dk.

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