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Journal of Bacteriology, May 1999, p. 3033-3038, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The A Modules of the Azotobacter vinelandii Mannuronan-C-5-Epimerase AlgE1 Are Sufficient for both Epimerization and Binding of Ca2+

Helga Ertesvåg* and Svein Valla

UNIGEN Center for Molecular Biology and Department of Biotechnology, Norwegian University of Technology and Science, N-7489 Trondheim, Norway

Received 3 October 1998/Accepted 9 March 1999

The industrially important polysaccharide alginate is composed of the two sugar monomers beta -D-mannuronic acid (M) and its epimer alpha -L-guluronic acid (G). In the bacterium Azotobacter vinelandii, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least five secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of one of the epimerases, AlgE1, is A1R1R2R3A2R4. This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this article we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showed that an A module alone is sufficient for epimerization and that A1 catalyzed the formation of contiguous stretches of G residues in the polymer, while A2 introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca2+ dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca2+ concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.

* Corresponding author. Mailing address: UNIGEN, Medisinsk teknisk senter, N-7489 Trondheim, Norway. Phone: 47 73598680. Fax: 47 73598705. E-mail: Helga.Ertesvag{at}unigen.ntnu.no.

Journal of Bacteriology, May 1999, p. 3033-3038, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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