Unraveling the molecular mechanism of polysaccharide lyases for efficient alginate degradation
José Pablo Rivas-Fernández, M. Paul Vuillemin, Bo Pilgaard, Leesa J. Klau, Folmer Fredslund, Charlotte Lund-Hanssen, Ditte Hededam Welner, Anne S. Meyer, Jens Preben Morth, Flora Meilleur, Finn L. Aachmann, Carme Rovira, Casper Wilkens
Abstract
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. Here, the authors characterise the syn-β-elimination mechanism of a mannuronic specific alginate lyase. An integrated approach utilising NMR, QM/MM simulations, X-ray and neutron crystallography reveals a single catalytic residue to be both the general acid and base during catalysis.