The molecular basis for alginate oligosaccharide cleavage by a Bacteroides ovatus Polysaccharide Lyase family 38

Tobias Tandrup*, José Pablo Rivas Fernandez, Mikkel Madsen, Mette Errebo Rønne, Agnes B. Petersen, Leesa J. Klau, Anne Tøndervik, Casper Wilkens, Finn L. Aachmann, Carme Rovira, Birte Svensson

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsResearchpeer-review

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Abstract

Alginate is an anionic linear polysaccharide and a major component of the cell wall of brown algae, that holds an important role in the marine biosphere. Alginate and alginate oligosaccharides (AOS) have several applications as viscosifiers, texturizers, and for encapsulation in food, pharma, and biomedicine industrial sectors. Alginate is organized in blocks of β-D-mannuronic acid (M) and α-L-guluronic acid (G) or alternating MG, giving the polysaccharide characteristic properties, which vary with the structure, molecular size, and M/G ratio. Alginate decomposition into AOSs is orchestrated by enzymes called alginate lyases, belonging to several polysaccharide lyase (PL) families. Similar for all alginate active PL families, catalysis is employed by a β-elimination mechanism of cleavage between uronic acid residues with the formation of an unsaturated uronic acid at the non-reducing chain end. The structural folds associated with alginate lyases so far are the parallel β-helix, the β-jelly roll, the (α/α)6-toroid, and the (α/α)6-toroid + antiparallel β-sheet.

Here, we present a PL38 from a novel Bacteroides ovatus strain (BoPL38) of the human gut microbiota, enabling bacterial growth on alginate [1]. BoPL38 is to our knowledge the first PL38 alginate lyase confirmed through biochemical characterization and was found active on all three types of AOS. We present a crystallographic analysis of complex structures obtained with the three AOSs, revealing an (α/α)7-barrel with an active site architecture capable of binding oligo-M, oligo-G, and oligo-MG saccharides through distortion. Using site- directed mutagenesis we uncover the molecular basis of substrate cleavage and through NMR spectroscopy we identify the formation of unsaturated chain ends and the mode-of-action. Finally, we present QM/MM simulations of the reaction coordinate, revealing mechanistic details for both M-M and G-G decomposition.

This work is supported by the Independent Research Fund Denmark | Natural Sciences, Novo Nordisk Foundation, the Norwegian Research Council, Carlsberg Foundation, DanScatt, and DTU. Diffraction data were collected at synchrotron radiation facilities MAXIV and ESRF.
Original languageEnglish
Title of host publicationDigitally Driven Biotechnology: 4th DTU Bioengineering symposium
Number of pages1
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Publication date2023
Pages45-45
Article number16
Publication statusPublished - 2023
Event4th DTU Bioengineering symposium - Kgs. Lyngby, Denmark
Duration: 26 Oct 202326 Oct 2023

Conference

Conference4th DTU Bioengineering symposium
Country/TerritoryDenmark
CityKgs. Lyngby
Period26/10/202326/10/2023

Keywords

  • Alginate lyase
  • Crystal structure
  • Substrate complexes
  • Enzyme mechanism

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