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OnderzoekGBBBiotransformation and BiocatalysisResearch

Halohydrin dehalogenase

D.B. Janssen
Biotransformation and Biocatalysis Group
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen

Halohydrin dehalogenase: discovery, engineering, and new biocatalytic applications

Dehalogenation of haloalcohols

Halohydrin dehalogenases are enzymes that convert vicinal halohydrins to an epoxide, a halide ion, and a proton. We have discovered three of these enzymes in bacteria that grow on halogenated compounds as a carbon source (Fig. 1A). The halohydrin dehalogenases remove halogen substituents in order to detoxify the compounds and allow them to enter central metabolism. During the biodegradation of epichlorohydrin, for example, the compound is first ring-opened by an epoxide hydrolase and the dehalogenated to form glycidol, an epoxide. In view of the fascinating properties of enzymes that cleave carbon-halogen bonds, we have studied halohydrin dehalogenases in detail, which started with cloning and expression of three different genes that encode these enzymes (van Hylckama Vlieg et al. 1989).

Fig. 1. Halohydrin dehalogenases. A) Bacteria that have catabolic pathways involving these enzymes. 1, Arthrobacter AD2 (HheA); 2, Mycobacterium GP1 (HheB), 3, Agrobacterium AD1 (HheC). B) Active site in HheC, showing proton abstraction with the negative charge that develops on the oxygen going to the halogen substituent, which is displaced. The halide-binding site is not indicated.
Fig. 1. Halohydrin dehalogenases. A) Bacteria that have catabolic pathways involving these enzymes. 1, Arthrobacter AD2 (HheA); 2, Mycobacterium GP1 (HheB), 3, Agrobacterium AD1 (HheC). B) Active site in HheC, showing proton abstraction with the negative charge that develops on the oxygen going to the halogen substituent, which is displaced. The halide-binding site is not indicated.

Catalytic mechanism

The sequence showed that halohydrin dehalogenases belong to the SDR (short-chain dehydrogenase-reductase) superfamily of proteins, which are oxidoreductases that use NAD or NADP as the cofactor. However, the halohydrin dehalogenase reaction is not a redox reaction, and in agreement with this, the dehalogenases do not have the typical Rossmann fold sequence motif that is conserved in the SDR enzymes, and they do not bind NAD(P). The three catalytic residues (Tyr, Ser and Arg (or Lys) that are involved in proton abstraction during alcohol oxidation are conserved; however, which led to a mechanism for HheC as is shown in Fig. 1B.

Fig. 2. Left: tetrameric structure of halohydrin dehalogenase. The yellow regions are the places where substrate enters the active sites. Right: close up of the halide-binding region. In this picture it is occupied by the azido-group (blue) of 1-p-nitrophenyl-2-azido-ethanol (green), which is bound in the active site. The catalytic triad residues are shown on top (pink). Bottom: the halide-binding loop (grey and marine) and a water molecule.
Fig. 2. Left: tetrameric structure of halohydrin dehalogenase. The yellow regions are the places where substrate enters the active sites. Right: close up of the halide-binding region. In this picture it is occupied by the azido-group (blue) of 1-p-nitrophenyl-2-azido-ethanol (green), which is bound in the active site. The catalytic triad residues are shown on top (pink). Bottom: the halide-binding loop (grey and marine) and a water molecule.

Detailed insight in the catalytic mechanism was obtained when the X-ray structure of HheC became available (de Jong et al., 2003, Tange et al., 2003) (Fig. 2). The enzymes indeed have a catalytic triad (or tetrad if we include an aspartic acid residue on the surface of the protein that may be involved in proton transfer to the solvent) that is involved in proton abstraction (Fig. 1B). There is also a halide binding site (Fig. 2B). Two structures are known (HheC, HheA) (de Jong et al., 2003, 2006).

Fig. 3. Biocatalytic reactions with halohydrin dehalogenase.Top: enantio­selective dehalogenation of haloalcohols; bottom: enantioselective ring opening of epoxides with different nucleophiles.
Fig. 3. Biocatalytic reactions with halohydrin dehalogenase.Top: enantio­selective dehalogenation of haloalcohols; bottom: enantioselective ring opening of epoxides with different nucleophiles.

Biocatalysis

The conversions catalyzed by halohydrin dehalogenases are reversible, depending on the nature of the leaving group. In the reverse (epoxide-ring opening) reaction various alternative negatively charged nucleophiles can be accepted, such as cyanide (Majeric et al., 2006) and azide (Lutje Spelberg et al., 2001). Thus a wide range of reactions can be carried out (Hasnaoui et al., 2008). An important property is the enantioselectivity of halohydrin dehalogenase. We have investigated:

- Kinetic resolution of halohydrins for preparation of enantiopure epoxides and (remaining) halohydrins. Tandem     reactions can be used to shift the equilibrium towards complete conversion (Lutje Spelberg et al., 1999, Haak et al., 2007).

- enantioselective azide-mediated ring opening of epoxides. A range of azidoalcohols derived from styrene oxide derivatives can be prepared (Lutje Spelberg et al., 2001)

- cyanide-mediated ring opening. Optically active cyanohydrins can be produced (Majeric et al., 2006)

- in case of 2,2-disubstituted epoxides, cyanide and azide-mediated ring opening gives enantiopure tertiary alcohols, which are not easily prepared in other ways (Majeric et al., 2007)

- tandem reactions, with ring closure of a halohydrin and ring-opening with cyanide. This reaction has also been explored by Codexis using mutants derived of HheC and can be used for the preparation of statin side chain building block (Majeric et al., 2007)

- dynamic resolution of epichloro- and epibromohydrin (Lutje Spelberg et al., 2004)

- nitrite-mediated ring opening of epoxides, can give nitroalcohol compounds and nitrite esters. The latter will hydrolyze spontaneously to produce a diol, and in this way the halohydrin dehalogenase effectively functions as an enantioselective epoxide hydrolase (Hasnaoui et al., 2005).

Thus, products of this enzymatic epoxide ring opening are enantiomerically pure epoxides or derivatives thereof which can be used as chiral building blocks for the production of a wide range of fine chemicals (Hasnaoui et al., 2008).

Protein engineering

On basis of the structure we have designed mutants with increased catalytic activity (Tang et al., 2005), enantioselectivity (Tang et al., 2003, 2005), and stability (Tang et al., 2002).

Fig. 4. Indicator agar plate assay for screening halohydrin dehalogenase activity of clones on plates. Left: active clones; right: inactive clones.
Fig. 4. Indicator agar plate assay for screening halohydrin dehalogenase activity of clones on plates. Left: active clones; right: inactive clones.

Further reading

de Jong R.M., Kalk K.H., Tang L., Janssen D.B., Dijkstra B.W. 2006. 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. J Bacteriol. 188:4051-6.

de Jong RM, Tiesinga JJ, Rozeboom HJ, Kalk KH, Tang L, Janssen DB, Dijkstra BW. 2003. 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. EMBO J. 22: 4933-44.

de Jong RM, Tiesinga JJ, Villa A, Tang L, Janssen DB, Dijkstra BW. 2005. Structural basis for the enantioselectivity of an epoxide ring opening reaction catalyzed by haloalcohol dehalogenase HheC. J Am Chem Soc 127:13338-43.

de Vries EJ, Janssen DB. 2003. Biocatalytic conversion of epoxides. Curr Opin Biotechnol 14:414-420.

Haak RM, Tarabiono C, Janssen DB, Minnaard AJ, de Vries JG, Feringa BL. 2007. Synthesis of enantiopure chloroalcohols by enzymatic kinetic resolution. Org Biomol Chem. 5:318-23.

Hasnaoui, G, Lutje Spelberg JH, de Vries E, Tang L, Hauer B, Janssen DB. 2005. Nitrite-mediated hydrolysis of epoxides catalyzed by halohydrin dehalogenase from Agrobacterium radiobacter AD1: a new tool for the kinetic resolution of epoxides. Tetr. Asymm. 16: 1685-1692.

Hasnaoui-Dijoux G,Majerić Elenkov M, Lutje Spelberg JH, Hauer B, Janssen DB. 2008. Catalytic promiscuity of halohydrin dehalogenase and its application in enantioselective epoxide ring opening. ChemBioChem 9:1048-51

Janssen DB, Majeric-Elenkov M, Hasnaoui G, Hauer B, Lutje Spelberg JH. 2006. Enantioselective formation and ring-opening of epoxides catalysed by halohydrin dehalogenases. Biochem Soc Trans. 34:291-5.

Lutje Spelberg JHL, Vlieg JETV, Bosma T, Kellogg RM, Janssen DB. 1999. A tandem enzyme reaction to produce optically active halohydrins, epoxides and diols. Tetr. Assymm. 15: 2863-2870.

Lutje Spelberg JH, van Hylckama Vlieg JE, Tang L, Janssen DB, Kellogg RM. 2001. Highly enantioselective and regioselective biocatalytic azidolysis of aromatic epoxides. Org Lett 3:41-3.

Lutje Spelberg JH, Tang L, van Gelder M, Kellogg RM, and Janssen DB. 2002. Exploration of the biocatalytic potential of a halohydrin dehalogenase using chromogenic substrates. Tetrahedron: Asymmetry 13: 1083–1089.

Lutje Spelberg, JH, Tang, L, Kellogg, RM, Janssen, DB. 2004. Enzymatic dynamic kinetic resolution of epihalohydrins. Tetr Asymm 15: 1095- 1102

Majeric Elenkov M, Tang L, Hauer B, Janssen DB. 2006. Sequential kinetic resolution catalyzed by halohydrin dehalogenase. Org Lett 8:4227-9.

Majeric Elenkov M, Hauer B, Janssen DB. 2007. One-pot biocatalytic synthesis of methyl (S)-4-chloro-3-hydroxybutanoate and methyl (S)-4-cyano-3-hydroxybutanoate. Submitted to Practical Methods in Biocatalysis and Biotransformations.

Majeric Elenkov M, Hoeffken W, Tang L, Hauer B, Janssena DB. 2007. Enzyme-catalyzed nucleophilic ring opening of epoxides for the preparation of enantiopure tertiary alcohols. Adv. Synth. Catal. 349: 2279 – 2285

Majeric-Elenkov M, Hauer B, Janssen DB. 2006. Enantioselective ring opening of epoxides with cyanide catalysed by halohydrin dehalogenases: a new approach tonon racemic β -hydroxynitriles. Adv. Synth. Catal. 348: 579-585.

Tang L, Lutje Spelberg JH, Fraaije MW, Janssen DB. 2003. Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter. Biochemistry 42:5378-86.

Tang L, Torres Pazmiño DE, Fraaije MW,de Jong RM, Dijkstra BW, and Janssen DB. 2005. Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site. Biochemistry 44: 6609-6618.

Tang L, van Merode AE, Lutje Spelberg JH, Fraaije MW, Janssen DB. 2003. Steady-state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the active site and halide-induced conformational change. Biochemistry 42:14057-65.

Tang L., van Hylckama Vlieg JET, Fraaije M, Janssen DB. 2002. Improved stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by replacement of cysteine residues. Enz Microb Technol 30: 251-258.

van den Heuvel RHH, Tahallah N, Kamerbeek NM, Fraaije MW, van Berkel WJH, Janssen DB, Heck AJR. 2005. Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase. J Biol Chem 280:32115-21

van Hylckama Vlieg JET, Tang L, Lutje Spelberg JH, Smilda T, Poelarends GJ, Bosma T, van Merode AE, Fraaije MW, Janssen DB. 2001. Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases. J Bacteriol 183: 5058-66.

Last modified:15 May 2014 12.10 p.m.