Mechanistic basis for understanding the dual activities of the bifunctionalAzotobacter vinelandiimannuronan C-5 epimerase and alginate lyase AlgE7

Abstract

Mannuronan C-5 epimerases and alginate lyases are important enzymes for tailoring of the functional properties of alginate. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes, like AlgE7 from Azotobacter vinelandii, can perform both reactions. These enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their dual activity is not understood. In this study, we investigate mechanistic determinants involved in the bifunctional alginate lyase and epimerase activity of AlgE7. Based on sequence analyses a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using well-defined alginate substrates, the preferential cleavage sites of AlgE7 are found to be M|XM and G|XM. From the study of variants, it was found that R148 is particularly important for the lyase activity of AlgE7, and we obtained pure epimerase variants. Furthermore, the results suggest a catalytic reaction mechanism, with H154 as the catalytic base and Y149 as the catalytic acid. This study opens for further applications of alginate epimerases and lyases, by providing a better understanding of the reaction mechanism and how the two enzyme reactions can be altered by changes in reaction conditions.