Mitigating product inhibition in 2′‐hydroxybiphenyl‐2‐sulfinate desulfinase (<scp>DszB</scp>) with synthetic glycosylation

The combustion of sulfur-rich crude oil is toxic to the environment, making the removal of sulfur impurities a priority for the sustainable use of liquid fuels. Biodesulfurization via the 4S pathway is a promising approach due to its C-S bond cleavage specificity and mild operating conditions. However, biodesulfurization is not economically viable due to the slow turnover of 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), an enzyme catalyzing the conversion of 2'-hydroxybiphenyl-2-sulfinate to 2-hydroxybiphenyl and sulfite. Previous studies have identified product inhibition as the limiting factor in DszB, whereby solvent-exposed protein loops obstruct the active site after substrate binding. This closed conformation is stabilized by hydrophobic interactions between the loops and the product. Here, we propose an artificial glycosylation strategy to mitigate product inhibition in DszB. We modeled glycated DszB in the apo, ligand-bound, and product-bound states with molecular dynamics based on the AMOEBA polarizable force field, and analyzed the chemical positioning of the reactant and product compared to the wild type (WT). We find that the addition of glucose on three Ser loop residues increases the interaction of the loops with water, overcoming the weaker product-loop interactions, and thereby enabling product release. Importantly, the enhanced flexibility of the loops was subtle enough to not heavily disrupt the chemical positioning of the reactant, which suggests that the rate acceleration would be similar to that of the WT.