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J. Biol. Chem., Vol. 283, Issue 35, 23819-23828, August 29, 2008

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Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii^*

Henriëtte J. Rozeboom¹, Tonje M. Bjerkan¹², Kor H. Kalk, Helga Ertesvåg, Synnøve Holtan, Finn L. Aachmann, Svein Valla³, and Bauke W. Dijkstra⁴

From the Laboratory of Biophysical Chemistry, GBB, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands and the Department of Biotechnology, The Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491 Trondheim, Norway

Alginate is a family of linear copolymers of (14)-linked β-D-mannuronic acid and its C-5 epimer -L-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic -helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp¹⁵², an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr¹⁴⁹, His¹⁵⁴, and Asp¹⁷⁸ as being essential for activity. Tyr¹⁴⁹ probably acts as the proton acceptor, whereas His¹⁵⁴ is the proton donor in the epimerization reaction.

Received for publication, May 29, 2008

^* This work was supported by a grant from the Norwegian Research Council. 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.

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

¹ Both authors contributed equally to the article.

² Present address: SINTEF Materials and Chemistry, Biotechnology, N-7465 Trondheim, Norway.

³ To whom correspondence may be addressed. E-mail: svein.valla{at}biotech.ntnu.no. ⁴To whom correspondence may be addressed. E-mail: b.w.dijkstra{at}rug.nl.

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