Unraveling the Molecular mechanism of Polysaccharide Lyases for Efficient Alginate Degradation

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 β-elimination reaction, with Tyr serving as both Brønsted 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.