Abstract
The industrially important polysaccharide alginate is composed of
the two sugar monomers beta-D-mannuronic acid (M) and its epimer
alpha-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 A1R1R2R3A2R4. 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 A1 catalyzed the formation of contiguous stretches of G residues
in the polymer, while A2 introduces single G residues. These differences
are predicted to strongly affect the physical and immunological properties
of the reaction product. The epimerization reaction is Ca2+ dependent,
and direct binding studies showed that both the A and R modules bind
this cation. The R modules appeared to reduce the Ca2+ concentration
needed for full activity and also stimulated the reaction rate when
positioned both N and C terminally.
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