Biology:Reductive dehalogenases

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Reductive dehaholagenses (EC 1.97.1.8) are a group of enzymes utilized in organohalide respiring bacteria.[1][2] These enzymes are mostly attached to the periplasmic side of the cytoplasmic membrane and play a central role in energy-conserving respiratory process for organohalide respiring bacteria by reducing organohalides. During such reductive dehalogenation reaction, organohalides are used as terminal electron acceptors. They catalyze the following general reactions:

R-X + 2 e− + 2 H+ → R-H + H-X          
X-RR-X + 2 e− + 2 H+ → R=R + 2X−

These membrane-associated enzymes have attracted great interest for the detoxification of organohalide pollutants. Organohalide pollution is a serious global environmental issue affecting soil and groundwater; and reductive dehalogenases offer a promising natural tool for bioremediation.

Structure and mechanism

Reductive dehalogenases are related to the cobamide (or vitamin B12) family of enzymes. They contain a cobalamin at its catalytic active site, where actual reductive reaction occurs. They also harbor iron− sulfur clusters that supply the reducing equivalents.[1][3] All membrane-associated dehalogenases harbor a N-terminal twin-arginine (TAT) signal sequence (RRXFXK), which is a conserved signal peptide for membrane protein translocation. Monomeric as well as dimeric forms were previously reported.

Enzymatic mechanism is still understudied; however, several studies reported various mechanisms involving an organocobalt adduct, a single-electron transfer, and a halogen–cobalt bond.[1]

Common reductive dehalogenases studied

Reductive dehalogenases from Dehalobacter species

Reductive dehalogenases from Dehalococcoides species

Reductive dehalogenases from Desulfitobacterium species

Production methods

Native enzymes

The examples are those that can dechlorinate chloroform (TmrA), PCE (PceA), TCE (TceA), and VC (VcrA).[2] Purification of such enzymes in native forms are reportedly difficult; however, a few such enzymes were purified to near homogeneity.[10][11] Ultracentrifugation, membrane solubilization and a series of liquid chromatography are the commonly employed techniques to the isolation and purification. A chloroform reducing dehalogenase is the latest reductive dehalogenase that was successfully produced and purified.[4]

Heterologous expressions

The researchers in the field had turned their interest to heterologous expression of the same enzymes due to difficulties in obtaining these enzymes in the native form. Only have recently a few recombinant reductive dehalogenases been functionally expressed, bringing the dehalogenase research into next levels.[12][7][3][5] Those successful efforts facilitate further investigations on their biochemical and structural properties.

The first membrane-associated respiratory reductive dehalogenase was heterologously expressed in a soluble and active form and purified using Bacillus megaterium.[5]

Uses in bioremediation

In recent years, research on reductive dehalogenases have attracted great interest from both academic and industrial researchers for their potential application in bioremediation of organohalide contamination.

References

  1. ↑ 1.0 1.1 1.2 Jugder, Bat-Erdene; Ertan, Haluk; Lee, Matthew; Manefield, Michael; Marquis, Christopher P. (2015). "Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides" (in English). Trends in Biotechnology 33 (10): 595–610. doi:10.1016/j.tibtech.2015.07.004. ISSN 0167-7799. PMID 26409778. 
  2. ↑ 2.0 2.1 Jugder, Bat-Erdene; Ertan, Haluk; Bohl, Susanne; Lee, Matthew; Marquis, Christopher P.; Manefield, Michael (2016). "Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation" (in English). Frontiers in Microbiology 7: 249. doi:10.3389/fmicb.2016.00249. ISSN 1664-302X. PMID 26973626. 
  3. ↑ 3.0 3.1 Quezada, C.P.; Payne, K.A.P.; Leys, D. (2014). "Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation". Nature 517 (7535): 513–516. doi:10.1038/nature13901. PMID 25327251. 
  4. ↑ 4.0 4.1 Jugder, Bat-Erdene; Bohl, Susanne; Lebhar, Helene; Healey, Robert D.; Manefield, Mike; Marquis, Christopher P.; Lee, Matthew (2017-06-20). "A bacterial chloroform reductive dehalogenase: purification and biochemical characterization" (in en). Microbial Biotechnology 10 (6): 1640–1648. doi:10.1111/1751-7915.12745. ISSN 1751-7915. PMID 28631300. 
  5. ↑ 5.0 5.1 5.2 Jugder, Bat-Erdene; Payne, Karl A. P.; Fisher, Karl; Bohl, Susanne; Lebhar, Helene; Manefield, Mike; Lee, Matthew; Leys, David et al. (2018-01-24). "Heterologous Production and Purification of a Functional Chloroform Reductive Dehalogenase". ACS Chemical Biology 13 (3): 548–552. doi:10.1021/acschembio.7b00846. ISSN 1554-8929. PMID 29363941. 
  6. ↑ Tang, S.; Edwards, E. A. (2013-03-11). "Identification of Dehalobacter reductive dehalogenases that catalyse dechlorination of chloroform, 1,1,1-trichloroethane and 1,1-dichloroethane". Philosophical Transactions of the Royal Society B: Biological Sciences 368 (1616): 20120318. doi:10.1098/rstb.2012.0318. ISSN 0962-8436. PMID 23479748. 
  7. ↑ 7.0 7.1 Parthasarathy, Anutthaman; Stich, Troy A.; Lohner, Svenja T.; Lesnefsky, Ann; Britt, R. David; Spormann, Alfred M. (2015-03-04). "Biochemical and EPR-Spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi Strain VS". Journal of the American Chemical Society 137 (10): 3525–3532. doi:10.1021/ja511653d. ISSN 0002-7863. PMID 25686300. 
  8. ↑ Wagner, A. Segler, L. Kleinsteuber, S. Sawers, G. Smidt, H. Lechner, U. (2013). Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi. OCLC 1018969275. 
  9. ↑ Suyama, A.; Yamashita, M.; Yoshino, S.; Furukawa, K. (2002-07-01). "Molecular Characterization of the PceA Reductive Dehalogenase of Desulfitobacterium sp. Strain Y51". Journal of Bacteriology 184 (13): 3419–3425. doi:10.1128/jb.184.13.3419-3425.2002. ISSN 0021-9193. PMID 12057934. 
  10. ↑ Neumann, Anke; Wohlfarth, Gert; Diekert, Gabriele (1996-07-12). "Purification and Characterization of Tetrachloroethene Reductive Dehalogenase fromDehalospirillum multivorans". Journal of Biological Chemistry 271 (28): 16515–16519. doi:10.1074/jbc.271.28.16515. ISSN 0021-9258. PMID 8663199. 
  11. ↑ Ni, S; Fredrickson, J K; Xun, L (1995). "Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1". Journal of Bacteriology 177 (17): 5135–5139. doi:10.1128/jb.177.17.5135-5139.1995. ISSN 0021-9193. PMID 7665493. 
  12. ↑ Mac Nelly, Anita; Kai, Marco; Svatoš, Aleš; Diekert, Gabriele; Schubert, Torsten (2014-05-09). "Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains". Applied and Environmental Microbiology 80 (14): 4313–4322. doi:10.1128/aem.00881-14. ISSN 0099-2240. PMID 24814779. Bibcode: 2014ApEnM..80.4313M. 



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