Biochemical analysis of the processive mechanism for epimerization of alginate by mannuronan C-5 epimerase AlgE4

Author:

CAMPA Cristiana1,HOLTAN Synnøve2,NILSEN Nadra2,BJERKAN Tonje M.2,STOKKE Bjørn T.3,SKJÅK-BRÆK Gudmund2

Affiliation:

1. Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy

2. Norwegian Biopolymer Laboratory, Department of Biotechnology, The University of Science and Technology NTNU, Trondheim, Norway

3. Department of Physics, The University of Science and Technology NTNU, Trondheim, Norway

Abstract

The enzymes mannuronan C-5 epimerases catalyse the in-chain epimerisation of β-D-mannuronic acid to α-L-guluronic acid in the last step of alginate biosynthesis. The recombinant C-5 epimerase AlgE4, encoded by the soil bacteria Azotobacter vinelandii and expressed in Escherichia coli, exhibits a non-random mode of action when acting on mannuronan and alginates of various monomeric compositions. The observed residue sequence has been suggested previously to be due to either a preferred attack or a processive mode of action. Based on methodologies involving specific degrading enzymes, NMR, electrospray ionisation mass spectrometry and capillary electrophoresis we show here that on average 10 residues are epimerised for each enzyme–substrate encounter. A subsite model for the enzyme is analysed by the same methodology using native and 13C-labelled mannuronan oligomers as substrate for the AlgE4 epimerase. A hexameric oligomer is the minimum size to accommodate activity. For hexa-, hepta- and octameric substrates the third M residue from the non-reducing end is epimerised first.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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