Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohy drin hydrogen-halide lyases, catalyze the nucleophilic displacement of a ha logen by a vicinal hydroxyl function in halohydrins to yield epoxides. Thre e novel bacterial genes encoding halohydrin dehalogenases were cloned and e xpressed in Escherichia coli, and the enzymes were shown to display remarka ble differences in substrate specificity. The halohydrin dehalogenase of Ag robacterium radiobacter strain AD1, designated HheC, was purified to homoge neity. The k(cat) and K-m values of this 28-kDa protein with 1,3-dichloro-2 -propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology sea rch as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, c atalytically important serine and tyrosine residues that are highly conserv ed in the SDR family are also present in HheC and other halohydrin dehaloge nases. The third essential catalytic residue in the SDR family, a lysine, i s replaced by an arginine in halohydrin dehalogenases. A site-directed muta genesis study, with HheC as a model enzyme, supports a mechanism for halohy drin dehalogenases in which the conserved Tyr145 acts as a catalytic base a nd Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the subst rate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not invol ve a covalent enzyme-substrate intermediate.