Engineered halohydrin dehalogenase mediates remote enantiocontrolled dehalogenative hydroxylation via an unconventional mechanism.

Expanding enzyme catalytic diversity is essential to unlocking the full potential of biocatalysis for green chemistry. Halohydrin dehalogenases are promiscuous biocatalysts that typically mediate the intramolecular dehalogenative cyclization of haloalcohols to form cyclic ethers, as well as the ring-opening of these O-heterocycles with various anionic nucleophiles. Here we report that engineered variants of the halohydrin dehalogenase HheC instead catalyze an intermolecular dehalogenative hydroxylation of ε-haloalcohols with remote enantiocontrol. Mechanistic investigations establish that an Asp-Arg dyad serves as the key catalytic motif responsible for this unconventional dehalogenation pathway. Through directed evolution, we further improve both the remote stereoselectivity and catalytic efficiency of the enzyme and systematically profile its substrate scope. The biocatalytic process is also successfully scaled to preparative and gram scales for the synthesis of chiral ε-haloalcohols and ε-diols. Our work uncovers an unusual dehalogenation mechanism and a practical platform for enantiocontrolled synthesis of chiral ε-substituted alcohols.