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

Abstract

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. 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 search 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, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and 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 substrate 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 involve a covalent enzyme-substrate intermediate.

FIG. 1

Sequence alignment of halohydrin dehalogenases with SDRs with known three-dimensional structures. The sequences were aligned by using the multiple alignment program ClustalW and are shown in decreasing sequence similarity to HheC. Amino acids that are identical in six or more sequences are depicted below the sequence. Positions at which six or more similar residues occur are indicated by an asterisk. The position of the G/A-G/A-X-X-G/A-X-G/A fingerprint typical of the Rossman fold in the SDR family is indicated by $ on top of the alignment. The proposed active site residues in halohydrin dehalogenases are indicated by #. The structural elements identified in three-dimensional structures of SDR family members are underlined. The first line below the sequence alignment shows the secondary structure elements and the nomenclature of 7α-hydroxysteroid dehydrogenase (29). β-Strands are indicated as double broken lines, and α-helices are indicated as single broken lines. The second, third, and fourth lines show the predicted secondary structure elements for HheC, HheA_AD2, and HheB_GP1, respectively. The nomenclature of the sequences is explained in Table 1.

FIG. 2

Proposed tertiary structure of halohydrin dehalogenase (HheC) of A. radiobacter AD1. A ribbon representation of residues Lys52 to Ile246 of HheC is shown. The active site residues that are critical for catalysis are indicated. Due to low sequence homology, the N-terminal fraction of HheC could not be predicted by using SDR proteins as a template.

FIG. 3

Proposed reaction mechanism of halohydrin dehalogenase. (A) Reaction mechanism and active site residues of SDRs exemplified by 7α-hydroxysteroid dehydrogenase of E. coli (adapted from references and 29). Interactions of the conserved lysine residue with the ribose moiety of the cofactor are not included. (B) Proposed reaction mechanism and active site residues of halohydrin dehalogenase exemplified by HheC of A. radiobacter (see text for explanation).