# Mode of Action of AlgE1: A Modular Mannuronate C‑5 Epimerase

## TL;DR

This study reveals how the AlgE1 enzyme modifies alginates by understanding its structure and function, which is important for industrial applications.

## Contribution

The study provides new insights into the mode of action and modular function of AlgE1, including the order of activity and effects of module rearrangement.

## Key findings

- Inactivating individual A-modules clarified their distinct roles in AlgE1's function.
- Switching the positions of A-modules increased initial reaction rates but reduced the formation of long G-blocks.
- AlgE1 processes its substrate starting with the C-terminal module.

## Abstract

The mannuronate C-5
epimerase AlgE1 from 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.