Halohydrin dehalogenase

A halohydrin dehalogenase is an enzyme involved in the bacterial degradation of vicinal halohydrins. In several species of bacteria, it catalyses the dehalogenation of halohydrins to produce the corresponding epoxides.[1] Different isoforms of the enzyme fall into one of three groups, A, B or C.[2] Halogenases of the same class are genetically similar, but differ greatly from halogenases from a different group.[2][3] Currently the most well-studied isoform is HheC which is purified from the bacterial species Agrobacterium radiobacter.[4] The ability to dehalogenate organic compounds as well as form enantiomeric selective epoxides have generated interest in the potential of this enzyme in the biochemical field.[5]

Structure

Currently of three known classes of halohydrin dehalogenases, only two have been described by x-ray crystallography studies.[6][7] However, both of these classes have similar structure which can be described as follows(1):[3] a halohydrin dehalogenase is structured as a tetramer with a symmetry characteristic of a dimer of dimers.[8] Each monomeric subunit consists of seven alpha helices and nine beta-sheets.[3] These monomers interact via the two longest alpha helices to form an alpha-helical bundle to form a dimer. The final quaternary structure is formed when two dimers interact via a different set of alpha helices and anti-parallel beta-sheets; interactions between the beta-sheets are thought to be a combination of both hydrophobic and electrostatic attraction.[8]

There is approximately one catalytic site per monomer subunit giving a total of four possible catalytic sites on the enzymatic tetramer. The active site consists of a Ser132-Tyr145-Arg149 catalytic triad.[3] The serine and tyrosine residues function to stabilize the substrate and its intermediate, while the arginine alters the pKa of Tyr145 to make it catalytically active.[8]

Mechanism

Halohydrin dehalogenases mechanistically cleaves the carbon-halogen bond through the formation of an epoxide from a vicinal hydroxyl group.[8][3] The substrate binds to the active site through hydrogen bonding that is coordinated by Ser132 and the deprotonated form of Tyr145. Failure to deprotonate Tyr145 by the Arg149 residue results in destabilization of the interaction between the enzyme and substrate resulting in reduced biological activity. The oxygen in Tyr145 deprotonates the hydroxyl group of the substrate. The deprotonated oxygen then acts as a nucleophile and performs a Sn2 reaction on the vicinal carbon that is bonded to the halogen; this releases a halogen ion and simultaneously forms an epoxide. Dehalogenases are also able to catalyze the ring-opening of the epoxide. The active site is large enough to accommodate a nucleophile which can perform a nucleophilic attack on the epoxide, opening the epoxide ring and adding a new functional group to the substrate.[8]

Overall mechanistic action of halohydrin dehalogenases

In regards to the geometry of the product, both class A and B dehalogenases have a low selective preference for the (S)-epoxide isomer.[9][10] However, the preference for the formation of the (R)-epoxide isomer catalyzed by enzymes in class C, particularly HHeC, is high. One study reports that HHeC catalyzed (R)-epoxide up to 99% enantiomeric excess.[8] However, the technology to purify this enzyme and utilize it on an industrial scale has yet to remain optimized.[11]

References
  1. Fauzi AM, Hardman DJ, Bull AT (1996). "Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria". Appl Microbiol Biotechnol. 46: 660–666. doi:10.1007/s002530050877.
  2. van Hylckama Vlieg JE, Tang LX, Lutje Spelberg JH, Smilda T, Poelarends GJ, Bosma T, van van Merode AE, Fraaije MW, Janssen DB (2001). "Halohydrin dehalogenases are structurally and mechanistically related to shortchain dehydrogenases/reductases". J Bacteriol. 183: 5058–5066. doi:10.1128/jb.183.17.5058-5066.2001.
  3. You ZY, Liu ZQ, Zheng YG (2013). "Properties and biotechnological applications of halohydrin dehalogenases: current state and future perspectives". Appl Microbiol Biotechnol. 97: 9–21. doi:10.1007/s00253-012-4523-0.
  4. http://www.rug.nl/research/biotransformation-biocatalysis/research/biodegr
  5. Choi WJ, Choi CY (2005). "Production of chiral epoxides: epoxide hydrolase-catalyzed enantioselective hydrolysis". Biotechnol Bioprocess. 10: 167–179. doi:10.1007/bf02932009.
  6. de Jong RM, Rozeboom HJ, Kalk KH, Tang LX, Janssen DB, Dijkstra BW (2002). "Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1". Acta Crystallogr D. 58: 176–178. doi:10.1107/s0907444901019618.
  7. de Jong RM, Kalk KH, Tang L, Janssen DB, Dijkstra BW (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: 4051–4056. doi:10.1128/jb.01866-05.
  8. 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: 4933–4944. doi:10.1093/emboj/cdg479. PMC 204463.
  9. Tang LX, Zhu XC, Zheng HY, Jiang RX, Elenkov MM (2012). "Key residues for controlling enantioselectivity of halohydrin dehalogenase from Arthrobacter sp. strain AD2, revealed by structure-guided directed evolution". Appl Environ Microbiol. 78: 4051–4056. doi:10.1128/AEM.06586-11. PMC 3318787. PMID 22327597.
  10. Elenkov MM, Hauer B, Janssen DB (2006). "Enantioselective ring opening of epoxides with cyanide catalysed by halohydrin dehalogenases: a new approach to non-racemic β-hydroxy nitriles". Advanced Synthesis & Catalysis. 348: 579–585. doi:10.1002/adsc.200505333.
  11. Assis HM, Sallis PJ, Bull AT, Hardman DJ (1998). "Biochemical characterization of a haloalcohol dehalogenase from Arthrobacter erithii H10a. Enzyme". Enzyme Microb Technol. 22: 568–574. doi:10.1016/s0141-0229(97)00254-8.
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