The A Modules of the Azotobacter vinelandii Mannuronan-C-5-Epimerase AlgE1 Are Sufficient for both Epimerization and Binding of Ca(2+)

The industrially important polysaccharide alginate is composed of the two sugar monomers β-d-mannuronic acid (M) and its epimer α-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 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 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 A(1) catalyzed the formation of contiguous stretches of G residues in the polymer, while A(2) introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca(2+) dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca(2+) concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.