Mode of Action of AlgE1: A Modular Mannuronate C-5 Epimerase

The mannuronate C-5 epimerase AlgE1 from Azotobacter vinelandii introduces long blocks of guluronate (G) into alginate. AlgE1 is an elongated enzyme consisting of six modules, of which two are catalytically active modules (A-modules). For industrial applications, G-rich alginates are sought after, and previous studies have shown that AlgE1 can be used for the valorization of both seaweed-derived and microbially produced alginates, but a complete understanding of the mode of action of AlgE1 is lacking. This study gives new data on the overall shape and conformational freedom of the AlgE1 enzyme in solution in the presence and absence of a substrate. With this basis, the questions of how the modules of AlgE1 work together and how the enzyme moves on the substrate have been addressed. The two A-modules were inactivated individually, which clarified the roles of each A-module and showed that small changes in the full-length construct affect the mode of action. The relative positions of the A-modules were switched, which resulted in two new enzymes with an initial reaction rate higher than that of the WT but with a reduced capacity to form long G-blocks. To understand the orientation of AlgE1 in processing of its substrate, lyase activity was introduced at different positions in AlgE1, and it could be concluded that AlgE1 processes the substrate with the C-terminal acting first. Overall, this study gives a completely new insight into the mode of action of AlgE1, which is important for further development and use of alginate epimerases in industrial applications.