The A modules of the Azotobacter vinelandii mannuronan-C-5-epimerase AlgE1are sufficient for both epimerization and binding of Ca2+

The industrially important polysaccharide alginate is composed of the two s ugar monomers beta-D-mannuronic acid (M) and its epimer alpha-L-guluronic a cid (G). In the bacterium Azotobacter vinelandii, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least fiv e secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 am ino acids). The modular structure of one of the epimerases, AlgE1, is A(1)R (1)R(2)R(3)A(2)R(4). This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this arti cle we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showe d that an A module alone is sufficient for epimerization and that A, cataly zed the formation of contiguous stretches of G residues in the polymer, whi le A(2) introduces single G residues. These differences are predicted to st rongly affect the physical and immunological properties of the reaction pro duct. The epimerization reaction is Ca2+ dependent, and direct binding stud ies showed that both the A and R modules bind this cation. The R modules ap peared to reduce the Ca2+ concentration needed for full activity and also s timulated the reaction rate when positioned both N and C terminally.