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 ...

Halo alcohol dehalogenase HheC catalyzes the highly enantioselective dehalogenation of vicinal halo alcohols to epoxides, as well as the reverse reaction, the enantioselective and beta-regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. To investigate this latter reaction, we determined X-ray structures of complexes of HheC with the favored and unfavored enantiomers of para-nitrostyrene oxide. The aromatic parts of the two enantiomers bind in a very similar way, but the epoxide ring of the unfavored (S)-enantiomer binds in a nonproductive inverted manner, with the epoxide oxygen and Cbeta atom positions interchanged with respect to those of the favored (R)-enantiomer. The calculated difference in relative Gibbs binding energy is in agreement with the observed loss of a single hydrogen bond in the S bound state with respect to the R bound state. Our results indicate that it is the nonproductive binding of the unfavored (S)-enantiomer, rather than the difference in affinity for the two enantiomers, that allows HheC to catalyze the azide-mediated ring opening of para-nitrostyrene oxide with high enantioselectivity. This work represents a rare opportunity to explain the enantioselectivity of an enzymatic reaction by comparison of crystallographic data on the binding of both the favored and unfavored enantiomers.

Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.

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