Identification of a Pivotal Residue for Determining the Block Structure-Forming Properties of Alginate C-5 Epimerases

ACS Omega
Annalucia StanisciFinn L Aachmann

Abstract

Alginate 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.

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Methods Mentioned

BETA
X-ray
nuclear magnetic resonance
NMR
ion exchange chromatography
two
new hybrid
two hybrid
three
new

Software Mentioned

SWISS
TopSpin
WEBnm
MODEL

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