ao9b04490_si_005.avi (121.48 kB)
Identification of a Pivotal Residue for Determining the Block Structure-Forming Properties of Alginate C‑5 Epimerases
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posted on 2020-02-24, 14:40 authored by Annalucia Stanisci, Anne Tøndervik, Margrethe Gaardløs, Anders Lervik, Gudmund Skjåk-Bræk, Håvard Sletta, Finn L. AachmannAlginate is a linear copolymer composed
of 1→4 linked β-d-mannuronic acid (M) and its
epimer α-l-guluronic
acid (G). The polysaccharide is first produced as homopolymeric mannuronan
and subsequently, at the polymer level, C-5 epimerases convert M residues
to G residues. The bacterium Azotobacter vinelandii encodes a family of seven secreted and calcium ion-dependent mannuronan
C-5 epimerases (AlgE1–AlgE7). These epimerases consist of two
types of structural modules: the A-modules, which contain the catalytic
site, and the R-modules, which influence activity through substrate
and calcium binding. In this study, we rationally designed new hybrid
mannuronan C-5 epimerases constituting the A-module from AlgE6 and
the R-module from AlgE4. This led to a better understanding of the
molecular mechanism determining differences in MG- and GG-block-forming
properties of the enzymes. A long loop with either tyrosine or phenylalanine
extruding from the β-helix of the enzyme proved essential in
defining the final alginate block structure, probably by affecting
substrate binding. Normal mode analysis of the A-module from AlgE6
supports the results.