Decoding Molecular Mechanism Underlying Human Olfactory Receptor OR8D1 Activation by Sotolone Enantiomers.

Sotolone, a chiral compound, plays an important role in the food industry. Herein, ( R )-/( S )-sotolone were separated to determine their odor characteristics and thresholds in air ( R -form: smoky, burned, herb, and green aroma, 0.0514 μg/m 3 ; S -form: sweet, milk, acid, and nutty aroma, 0.0048 μg/m 3 ). OR8D1 responses to ( R )-/( S )-sotolone were detected in a HEK293 cell-based luminescence assay. ( S )-Sotolone was a more potent agonist than ( R )-sotolone (EC 50 values of 84.98 ± 1.05 and 167.20 ± 0.25 μmol/L, respectively). Molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area analyses confirmed that the combination of ( S )-sotolone and OR8D1 was more stable than that of ( R )-sotolone. Odorant docking, multiple sequence alignments, site-directed mutagenesis, and functional studies with recombinant odorant receptors (ORs) in a cell-based luminescence assay identified 11 amino-acid residues that influence the enantioselectivity of OR8D1 toward sotolone significantly and that N206 5.46 was indispensable to the activation of OR8D1 by ( S )-sotolone.