Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

Abstract

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)Hdependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the ®rst structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors. Keywords: haloalcohol dehalogenase/SDR family/shortchain dehydrogenase/X-ray structure IntroductionDehalogenases are enzymes that are able to cleave carbonhalogen bonds . Structural characterization of haloalkane dehalogenases and haloacid dehalogenases demonstrated that these hydrolytic enzymes are evolutionarily related to widespread esterase and phosphatase families (Ollis et al., 1992;Hisano et al., 1996;Ridder and Dijkstra, 1999). Haloalcohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen-halide lyases, cannot be classi®ed in these existing dehalogenase families. Instead, they show low sequence similarity to members of the short-chain dehydrogenase/reductase (SDR) family . This family contains redox enzymes that depend on NAD(P)H, which is bound in a characteristic dinucleotide binding fold (Rossmann fold) (Rossmann et al., 1974). They have a conserved catalytic triad of Ser, Tyr and Lys/Arg residues (Jo Èrnvall et al., 1995;Oppermann et al., 2003), which is also present in the haloalcohol dehalogenases . Many structures of shortchain dehydrogenases/reductases in complex with dinucleotides and substrates have revealed the structural details of the reactions catalyzed by them (Jo Èrnvall et al., 1995;Filling et al., 2002;Oppermann et al., 2003). In addition, the structure of a dinucleotide-binding transcription factor that lacked the catalytic tyrosine and thus oxidoreductase activity showed that the SDR fold also functions in nonenzymatic activities (Stammers et al., 2001).Haloalcohol dehalogenases catalyze the intramolecular displacement of a halogen by the vicinal hydroxyl group in 1,3-dichloro-2-propanol, yielding its ...

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

“…Molecular replacement solutions were obtained with the program AMoRe available in CCP4 (2, 10), using diffraction data between 8-and 4-Å resolution. As a search model, the atomic coordinates of the tetrameric HheC from A. radiobacter AD1 (PDB code 1PWX [4]) were used. Rigid body refinement of the solutions gave a starting correlation coefficient of 0.73 and an R factor of 32.2%.…”

“…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.…”

“…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. 1B) (4). The halogen atom is bound in a spacious halide-binding site, which also stabilizes the halide product.…”

Investigation of Halohydrins Degradation by Whole Cells and Cell-free Extract of Pseudomonas putida DSM 437: A Kinetic Approach

“…The retention time of glycerol was 10.303 min. The degradation of 1,3-DCP is considered to follow the proposed metabolic pathway ( Figure 3) 6, 15,26 . The first reaction of the pathway is supposed to be the dehalogenation of 1,3-DCP to epichlorohydrin by a haloalcohol dehalogenase (also also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyase, HDDH).…”

“…The first reaction of the pathway is supposed to be the dehalogenation of 1,3-DCP to epichlorohydrin by a haloalcohol dehalogenase (also also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyase, HDDH). Haloalcohol dehalogenases catalyze the intramolecular displacement of a halogen by the vicinal hydroxyl group in 1,3-DCP, yielding its corresponding epoxide, a halide ion, and a proton without the need of cofactors [26][27] . In concert with a well characterized epoxide hydrolase, the haloalcohol dehalogenase constitutes an efficient degradation pathway of 1,3-DCP and epichlorohydrin into non-toxic metabolites.…”