Mechanism and Origin of Stereoselectivity of Halohydrin Dehalogenase-Catalyzed Ring-Expansion Reaction of Spiro-Epoxides

Abstract The detailed mechanism of halohydrin dehalogenase (HHDH)-catalyzed ring-expansion reaction between spiro-epoxides and the nucleophile OCN– to form spiro-oxazolidinones was investigated using molecular docking, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations. Molecular docking and molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) results revealed that the R-configurational substrate exhibited significantly superior binding affinity compared to the S-configurational one, validating the stable binding mode within the enzyme′s active pocket. The fundamental pathway initiates with the nucleophilic attack of OCN– accompanied by proton transfer, which is followed by ring closure coupled with a proton transfer to form a five-membered ring. A total of four possible selective nucleophilic attack pathways were evaluated, and the most energetically favorable pathway associated with the R-configurational product has an energy barrier of 17.1 kcal/mol, which is close to the experimental value of 18.1 kcal/mol derived from kinetic parameters. This study provides valuable theoretical insights for the future engineering of HHDHs for chiral spiro-oxazolidinone synthesis.