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<p>Alginate lyases (ALs) catalyze the depolymerization of brown macroalgae alginates, widely used naturally occurring polysaccharides. Their molecular reaction&#xa0;mechanism remains elusive due to the lack of catalytically competent Michaelis-Menten-like complex structures. Here, we provide structural snapshots 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 <i>syn</i> β-elimination reaction, with Tyr serving as both Brønsted base and acid, involving a carbanion-type transition state. This study not only reconciles previous structural and kinetic discrepancies, but also establishes a comprehensive PL reaction mechanism which is most likely&#xa0;applicable across&#xa0;all enzymes of the PL7 family as well as other PL families.</p>

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Unraveling the molecular mechanism of polysaccharide lyases for efficient alginate degradation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月18日

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  • José Pablo Rivas-Fernández,
  • Marlene Vuillemin,
  • Bo Pilgaard,
  • Leesa J. Klau,
  • Folmer Fredslund,
  • Charlotte Lund-Hanssen,
  • Ditte H. Welner,
  • Anne S. Meyer,
  • J. Preben Morth,
  • Flora Meilleur,
  • Finn L. Aachmann,
  • Carme Rovira,
  • Casper Wilkens

摘要

Alginate lyases (ALs) catalyze the depolymerization of brown macroalgae alginates, widely used naturally occurring polysaccharides. Their molecular reaction mechanism remains elusive due to the lack of catalytically competent Michaelis-Menten-like complex structures. Here, we provide structural snapshots 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 β-elimination reaction, with Tyr serving as both Brønsted base and acid, involving a carbanion-type transition state. This study not only reconciles previous structural and kinetic discrepancies, but also establishes a comprehensive PL reaction mechanism which is most likely applicable across all enzymes of the PL7 family as well as other PL families.