Halohydrin dehalogenase

Scheme 10.33 Schematic representation of the mechanism of SDRs (top) and the halohydrin dehalogenase HheC (bottom). Scheme adapted from Reference 129.

The crystal structure of the halohydrin dehalogenase from the soil bacterium Agrobacterium radiobacter ADI, HheC, has been solved [129]. HheC is structurally related to the family of NAD(P)H-dependent short-chain dehydrogenases/reduc-... 

The reversibility of halohydrin dehalogenase-catalyzed reactions has been used for the regioselective epoxide-opening with nonnatural nucleophiles (an example is given in Scheme 10.34) [133]. The stereoselectivity of the enzyme results in the resolution of the racemic substrate. At the same time, the regioselectivity imposed by the active site geometry yields the anti-Markovnikov product. [128]... 

Figure 6.74 Ring-opening reactions catalyzed by halohydrin dehalogenase.

Ring opening of epoxides with nucleophiles other than water (Cl, BrT I, NOz , CN ) can also be catalyzed by halohydrin dehalogenase enzymes (EC 3.8.1.5, also named haloalkane dehalogenase or haloalcohd dehalogenase) (Figure 6.74) [197]. 

Procedure 1 Expression and Purification of a Mutated Halohydrin Dehalogenase HheC-W249F... 

Halohydrin dehalogenase activity was determined by monitoring halide liberation at 30 °C in tris-S04 buffer (50 mM, pH 8.0) containing 5 mM 1,3-dichloropropanol or 1,3-dibromopropanol as the substrate. All buffers used for activity assay were prepared with bidest water. From the incubation mixture, 0.5 ml samples were taken and mixed with 1.6 ml of H2O, 0.2 ml or halide reagent 1 and 0.2 ml of halide reagent II. Absorbances were read at 460 nm. A calibration curve of 0-1 mM of chloride or bromide was used to calculate the concentration of halide. The extinctions at 460 nm should be below 0.4 (for chloride) or 0.8 (for bromide). 

HheC-W249F (15 mg purified mutant halohydrin dehalogenase in 3 ml buffer)... 

To a solution of methyl 4-chloro-3-hydroxybutanoate (0.50 g, 3.3 mmol) in 62 ml tris-SO4 buffer (0.5 m, pH 7.5) NaCN was added (322 mg, 6.6 mmol), followed by addition of purified HheC-W249F halohydrin dehalogenase (15 mg in 3 ml buffer). The resulting mixture was stirred at ambient temperature (22 °C) for 5 h. 

Tang, L., Torres Pazmino, D.E., Fraaije, M.W., de Jong, R.M., Dijkstra, B.W. and Janssen, D.B., Improved catalytic properties of halohydrin dehalogenase hy modification of the halide-binding site. Biochemistry, 2005, 44, 6609. 

Majeric Elenkov, M., Tang, L., Hauer, B. and Janssen, D.B., Sequential kinetic resolution catalyzed by halohydrin dehalogenase. Org. Lett., 2006, 8, 4227. 

Figure 6.2 Overview of biocatalytic routes to vastatin side chains. PLE pig-liver esterase, ADH alcohol dehydrogenase, HHDH halohydrin dehalogenase, DERA 2-deoxy-D-ribose 5-phosphate aldolase.

Recently it was described that the halohydrin dehalogenases can be employed for the enantioselective formation of a new carbon-carbon bond. Their natural task is the dehalogenation of halohydrins, however this reaction can be reversed. When utilizing cyanide instead of a halide as the nucleophile in the reverse reaction the desired /ttiydroxynitrile is formed with good to excellent enantioselectivity (Scheme 5.34) [60]. 

Scheme 5.34 Application of a halohydrin dehalogenase for the synthesis of a carbon-carbon bond.

Halohydrin dehalogenases Halohydrins, epoxides Diols, epoxides, 3-hydroxynitriles 43... 

Suzuki T, Kasai N, Yamamoto R, Minamiura N. A novel generation of optically active 1,2-diols from the racemates by using halohydrin dehalogenase. Tetrahedron Asymmetry 1994 5 239-246. 

As an alternative to the enzymatic hydrolysis of epoxides, nonracemic vicinal diols may be obtained from epoxides via the nucleophilic ring-opening by nitrite catalyzed by halohydrin dehalogenase (a lyase). The corresponding nitrite-monoesters are spontaneously hydrolyzed to yield diols. For the application of this technique see Sect. 2.7.2. 

Previously, halohydrin dehalogenases were also termed haloalcohol dehalogenases or halohydrin epoxidases , see [1828]. 

First hints on the stereoselectivity of halohydrin dehalogenases were obtained from studies on the desymmetrization of prochiral 1,3-dichloropropan-2-ol yielding epichlorohydrin using resting cells of Corynebacterium sp. (Scheme 2.235) [1836]. In two-step sequence, (/ )-3-chloropropane-l,2-diol was formed in 74% e.e. via epichlorohydrin through the sequential action of an (unspecified) halohydrin de-halogenase and an epoxide hydrolase [1837]. Further studies revealed that these activities are widespread among bacteria [1838-1842]. 

---