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Originally published In Press as doi:10.1074/jbc.M102562200 on June 4, 2001

J. Biol. Chem., Vol. 276, Issue 34, 31542-31550, August 24, 2001
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The Catalytic Activities of the Bifunctional Azotobacter vinelandii Mannuronan C-5-Epimerase and Alginate Lyase AlgE7 Probably Originate from the Same Active Site in the Enzyme*

Britt Iren Glærum SvanemDagger , Wenche Iren StrandDagger , Helga ErtesvågDagger , Gudmund Skjåk-BrækDagger , Martin HartmannDagger , Tristan Barbeyron§, and Svein VallaDagger

From the Dagger  Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway and § Centre d'Etudes d'Océanologie et de Biologie Marine, CNRS, F-29680 Roscoff, France

The Azotobacter vinelandii genome encodes a family of seven secreted Ca2+-dependent epimerases (AlgE1-7) catalyzing the polymer level epimerization of beta -D-mannuronic acid (M) to alpha -L-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1-7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca2+-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly Gdown-arrow MM and/or Gdown-arrow GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.

* This work was supported by grants from the Norwegian Research Council, FMC Biopolymers AS, and by European Union Grant QLK3-1999-00034.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 47-73593320; Fax: 47-73591283; E-mail: svein.valla@chembio.ntnu.no.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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