Abstract
The alginate-degrading enzyme BoPL38 of the human gut bacterium Bacteroides ovatus 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 BoPL38 to act across all alginate structures. We resolved in crystallo complexes of BoPL38 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 syn- and anti-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 BoPL38 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.