The Swiss Army Knife of Alginate Metabolism: Mechanistic Analysis of a Mixed-Function Polysaccharide Lyase/Epimerase of the Human Gut Microbiota.

The alginate-degrading enzyme PL38 of the human gut bacterium CP926 degrades all three polysaccharide structures found in alginate, a major constituent of brown macroalgae, making it a valuable tool for the selective production of alginate oligosaccharides with industrial and biotechnological potential. Despite its abundance, alginate's heterogeneous composition limits its full utilization. Modification by epimerases and lyases can help to overcome this limitation, but typically requires distinct enzymes for each polysaccharide structure. Here, we combined experimental and computational approaches to elucidate the catalytic machinery that enables PL38 to act across all alginate structures. We resolved complexes of PL38 with alginate oligosaccharides, providing key insights into substrate binding. These structures informed QM/MM MD simulations, which uncovered distinct conformational and reaction pathways for mannuronate and guluronate conversion. The simulations identified different transition states, showing how a single active site architecture facilitates C5 proton abstraction at subsite +1 by Y298 and H243, enabling - and -β-elimination, respectively. A well-defined residue network mediates substrate recognition, and site-directed mutagenesis revealed that disruption of this network destabilizes the active site architecture. Notably, R292 plays a critical role in distorting the sugar at subsite +1 into a preactivated conformation while also stabilizing the active site tunnel through a salt bridge. Finally, NMR spectroscopy revealed that PL38 also catalyzes mannuronate-to-guluronate epimerization, highlighting its multifunctionality. These findings provide molecular insight into how a single enzyme accommodates alginate's structural diversity and offer new opportunities for enzymatic polysaccharide engineering.