ACS Chem Biol. 2026 Apr 20. doi: 10.1021/acschembio.5c00763. Online ahead of print.

ABSTRACT

Uncovering enzymes with promiscuous catalytic activity is significant in biocatalysis to expand their synthetic application toward sustainable chemical transformations, find non-native catalytic activity, and understand enzyme evolution. Mining the whole genome of organophosphate-degrading Sphingopyxis sp. MG to identify potential organochloride-degrading enzymes revealed the existence of two haloalkane dehalogenases (Hld), SphHldA and SphHldB. Both contain key active site residues typically seen in haloalkane dehalogenase-I (HLD-I) subfamily of enzymes. The SphHldB also possessed the active site residues of a typical epoxide hydrolase (EH) but showed no phylogenetic relationship with any of the typical HLD-I subfamily enzymes. Like in EHs with promiscuous Hld activity, the SphHldB has a second halide binding residue (Trp206) and a long α₄ helix. SphHldA displayed reasonable Hld activity with 13 structurally diverse halogenated substrates (up to 1.04 μmol min^-1 mg^-1), while SphHldB showed very low specific activity. Notably, SphHldA (984 nmol min^-1 mg^-1) exhibited >600-fold higher activity than LinB (∼1.62 nmol min^-1 mg^-1) toward the dehalogenation of the recalcitrant substrate 1,2-dichloropropane. Supporting the bioinformatics predictions, SphHldB exhibited EH activity both on aliphatic and aromatic epoxides, the highest up to 1.43 μmol min^-1 mg^-1 with 1-epoxyhexane. Its Hld activity toward a homologous carbon frame of 1-bromohexane was found to be below the detection limit (5.4 nmol min^-1 mg^-1), accounting for a > 260-fold higher EH/Hld-specific activity. SphHldB showed enantioselective hydrolysis of styrene epoxide, albeit with poor ee and insufficient regioselectivity, resulting in up to 100% yield with only 23% ee of the corresponding (R)-diol. These results suggest that while both SphHldA and SphHldB exhibit Hld activity, SphHldB has dual EH and Hld promiscuous catalytic activity in its native form. It seems to be a generalist epoxide hydrolase on its way to transit to a specialist Hld.

PMID:42008633 | DOI:10.1021/acschembio.5c00763