(575g) Conformational Changes of 2’-Hydroxybiphenyl-2-Sulfinate Desulfinase

Biodesulfurization using the 4S enzymatic pathway can effectively remove sulfur from refractory thiophenic molecules at ambient conditions. 2’-hydroxybiphenyl-2-sulfinate (HBPS) desulfinase (DszB), one of four enzymes in the 4S pathway, catalyzes the cleavage of the carbon-sulfur bond to produce 2-hydroxybiphenyl (HBP) and sulfite; this final step in the 4S pathway exhibits the slowest reaction rate, partially as a result of product inhibition. Here, we examine the effect of solvent on a large-scale conformational change, which may have implications for understanding the mechanism of product inhibition. Molecular Dynamics (MD) simulation was used to analyze how the HBPS substrate and inhibitory product, HBP, bind in DszB active site and influence protein conformational changes in two solutions: aqueous buffer and a biphasic hexane-water (0.5 vol/vol) solution. Three loops, including residues 50-60 (loop 1), 135-150 (loop 2), and 180-200 (loop 3), were identified as participating in the dominant conformational change; they are located close to active site and appear to form a “gate” that may be responsible for ligand ingress/egress. In aqueous solution, both HBPS and HBP remained bound in a stable position in the active site. HBPS-bound DszB displayed large fluctuations in a region distal to the active site (residues 220-240), and the entire protein backbone was less stable in comparison to HBP-bound DszB. Structural studies suggest the catalytic residues, Cys27, His60, and Arg70, remain close to the HBPS substrate and maintain substrate interactions; though in the HBP-bound DszB, the catalytic cysteine moved away from the active site along the simulation. In biphasic solution, HBPS-bound DszB showed improved rigidity distal from catalytic center. Due to the high affinity of HBP for the organic phase, HBP escaped the active site to the hexane-aggregated phase at the nonpolar DszB surface region, leaving the gate open facilitating the next reaction. Umbrella sampling provided a quantitative assessment of the energetic penalty for the gate conformational change. The energy cost associated with the gate opening from the close position was 3 kcal/mol. This value was independent of bound ligand and solvent conditions.