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.Halogenated aliphatics constitute an important class of environmental pollutants. Various microorganisms have evolved that are able to degrade some of these compounds and use them as sole sources of carbon and energy. Such organisms are of importance for bioremediation of polluted soil, groundwater, and wastewater. In most cases, specialized enzymes, designated dehalogenases, catalyze the cleavage of the carbonhalogen bonds, which is a key detoxification reaction. Hydrolytic dehalogenases have been studied extensively, which has resulted in detailed insight into the structure and mechanism of several enzymes of this class (8,33). For other dehalogenases, structural and mechanistic data are hardly available.Halohydrin dehalogenases, also referred to as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, occur in the degradation pathways of halopropanols and 1,2-dibromoethane, where they catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, yielding an epoxide, a proton, and a halide ion (7,22,30,31). These enzymes also efficiently catalyze the reverse reaction, the halogenation of epoxides, and the dehalogenation of vicinal chlorocarbonyls to hydroxycarbonyls (2, 14, 31). The interest in halohydrin dehalogenases increased when i...

The X-Ray Structure of the Haloalcohol Dehalogenase HheA from Arthrobacter sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family

“…Their sequences also indicated that they all are evolutionarily related to the large family of NAD(P)(H)-dependent short-chain dehydrogenases/reductases (SDRs) (21), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze various alcohol-ketone conversions (11). Crystallographic analysis of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 revealed that the tyrosine residue of the conserved Ser-Tyr-Arg catalytic triad is in a position to activate the hydroxyl group of the haloalcohol function for nucleophilic attack on the vicinal halogen-bearing carbon atom (Fig.…”

“…Over the years, detailed structural information has become available for several dehalogenases belonging to evolutionary unrelated families (3). One such family, which was recently characterized, comprises haloalcohol dehalogenases (4,18,21). The enzymes from this family were isolated from bacteria that are able to grow on vicinal haloalcohols, like 1,3-dichloro-2-propanol and 2,3-dichloro-1-propanol, or compounds that are degraded via haloalcohols, some of which are notable environmental pollutants.…”

“…Until now, six different haloalcohol dehalogenases, which can be grouped into the three subtypes (A, B, and C) on the basis of amino acid sequence similarities, have been isolated (21). Their sequences also indicated that they all are evolutionarily related to the large family of NAD(P)(H)-dependent short-chain dehydrogenases/reductases (SDRs) (21), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze various alcohol-ketone conversions (11).…”

Isolation and Characterization of a 1,3-Dichloro-2-Propanol-Degrading Bacterium

“…Enzymatic characteristics and activity against various haloalcohols of Deh-PY1 are compared with enzymes reported by other authors in Tables 3 and 4. Deh-PY1 was obtained from a bacterium of the genus Arthrobacter similar to AD2 9,22) and DehA, 6) but their characteristics differ. Deh-PY1 and AD2 are homo tetramer and dimer, respectively, whereas DehA is a hetero polymer.…”

Biocatalytic conversion of epoxides

“…These enzymes play a role in the biodegradation of some xenobiotic halogenated compounds. In Figure 1c, the proposed mechanism of the reversible ring opening is shown, which is based on the sequence similarity with the short-chain dehydrogenase/reductase (SDR) family of proteins [34 ]. The process involves activation of the epoxide by hydrogen bonding to a tyrosine.…”