Unraveling the Molecular mechanism of Polysaccharide Lyases for Efficient Alginate Degradation

Rivas-Fernandez, J. P.; Vuillemin, M.; Pilgaard, B.; J. Klau, L.; Fredslund, F.; Lund-Hanssen, C.; H. Welner, D.; S. Meyer, A.; Morth, J. P.; Meilleur, F.; L. Aachmann, F.; Rovira, C.; Wilkens, C.

doi:10.1101/2024.08.02.606366

bioRxiv · 10.1101/2024.08.02.606366

Unraveling the Molecular mechanism of Polysaccharide Lyases for Efficient Alginate Degradation

Rivas-Fernandez, J. P.·Vuillemin, M.·Pilgaard, B.·J. Klau, L.·Fredslund, F.·Lund-Hanssen, C.·H. Welner, D.·S. Meyer, A.·Morth, J. P.·Meilleur, F.·L. Aachmann, F.·Rovira, C.·Wilkens, C.

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Abstract

Alginate lyases (ALs) are essential for breaking down brown macroalgae alginates, widely used naturally-occurring polysaccharides. Their molecular mechanisms remain challenging due to the lack of catalytically competent Michaelis-Menten complex structures. We here provide structural snap-shots and dissect the mechanism of mannuronan-specific ALs from family 7 polysaccharide lyases (PL7), employing time-resolved NMR, X-ray, neutron crystallography, and QM/MM simulations. We reveal the protonation state of critical active site residues, enabling atomic-level analysis of the reaction coordinate. Our approach reveals an endolytic and asynchronous syn {beta}-elimination reaction, with Tyr serving as both Bronsted base and acid, involving a carbanion-type of transition state. This study not only reconciles previous structural and kinetic discrepancies, but also establishes a comprehensive PL reaction mechanism applicable across lyase families, which can guide the engineering of ALs for tailored alginate oligosaccharide production.

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Rivas-Fernandez, J. P., Vuillemin, M., Pilgaard, B., J. Klau, L., Fredslund, F., Lund-Hanssen, C., H. Welner, D., S. Meyer, A., Morth, J. P., Meilleur, F., L. Aachmann, F., Rovira, C., Wilkens, C.. 2024-08-03. Unraveling the Molecular mechanism of Polysaccharide Lyases for Efficient Alginate Degradation. https://doi.org/10.1101/2024.08.02.606366

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