Mechanistic Basis for Understanding the Dual Activities of the Bifunctional Azotobacter vinelandii mannuronan C-5-Epimerase and Alginate Lyase AlgE7

Margrethe Gaardløs, Tonje Marita Bjerkan Heggeset, Anne Tøndervik, David Tezé, Birte Svensson, Helga Ertesvåg, Håvard Sletta, Finn Lillelund Aachmann*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the monomer composition by catalysing the epimerization of β-d-mannuronic acid residues (M) into α-l-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which catalyse a β-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activity where H154 functions as the catalytic base and Y149 as the catalytic acid. Importance Post-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.
Original languageEnglish
Article numberAEM0183621
JournalApplied and Environmental Microbiology
Volume88
Issue number3
Number of pages18
ISSN0099-2240
DOIs
Publication statusPublished - 2022

Keywords

  • Alginate
  • Alginate C-5 epimerase
  • Alginate lyase
  • Multifunctional enzyme
  • Site-directed mutagenesis
  • Nuclear magnetic resonance (NMR)
  • Time-resolved NMR
  • Enzyme mechanism

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