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/m³; S-form: sweet, milk, acid, and nutty aroma, 0.0048 μg/m³). 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 (EC₅₀ 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.