Genome Mining of Sphingopyxis sp. MG Uncovers an Enzyme with Dual Catalytic Activity of Haloalkane Dehalogenase and Epoxide Hydrolase

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.