Fredslund, F. | bioRxiv Science

bioRxiv2024

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 {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.

molecular biology↗

bioRxiv2023

Sucrose phosphorylase from Alteromonas mediterranea: structural insight into the regioselective α-glucosylation of (+)-catechin

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea (AmSP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, AmSP-Q353F and AmSP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89% of a novel compound (+)-catechin-4'-O--D-glucopyranoside (CAT-4) for AmSP-P140D and 92% of (+)-catechin-3'-O--D-glucopyranoside (CAT-3) for AmSP-Q353F. The compound CAT-4 was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: AmSP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4 while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3.

biochemistry↗