Functional Expression and Characterization of Tetrachloroethene Dehalogenase From Geobacter sp.

Reductive dehalogenase (RDase) consists of two parts, RdhA and RdhB. RdhA is the catalytic subunit, harboring a cobalamin cofactor and two Fe–S clusters. RdhA is anchored to the cytoplasmic membrane via the membrane anchoring subunit, RdhB. There are many genes encoding RDases in the genome of organohalide-respiring bacteria, including Dehalococcoides spp. However, most genes have not been functionally characterized. Biochemical studies on RDases have been hampered by difficulties encountered in their expression and purification. In this study, we have expressed, purified and characterized RdhA of RDase for tetrachloroethene (PceA) from Geobacter sp. PceA was expressed as a fusion protein with a trigger factor tag in Escherichia coli. PceA was purified and denatured in aerobic condition. Subsequently, this protein was refolded in the presence of FeCl3, Na2S and cobalamin in anaerobic condition. The reconstituted PceA exhibited dechlorination ability for tetrachloroethene. UV-Vis spectroscopy has shown that it contains cobalamin and Fe-S clusters. Since this method requires anaerobic manipulation only in the reconstituting process and has a relatively high yield, it will enable further biochemical studies of RDases.

[1]  H. Ertan,et al.  Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation , 2016, Front. Microbiol..

[2]  Patrick K. H. Lee,et al.  A comparative genomics and reductive dehalogenase gene transcription study of two chloroethene-respiring bacteria, Dehalococcoides mccartyi strains MB and 11a , 2015, Scientific Reports.

[3]  H. Ertan,et al.  Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides. , 2015, Trends in biotechnology.

[4]  A. Spormann,et al.  Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS. , 2015, Journal of the American Chemical Society.

[5]  K. Fisher,et al.  Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation , 2014, Nature.

[6]  A. Svatoš,et al.  Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains , 2014, Applied and Environmental Microbiology.

[7]  S. Zinder,et al.  Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide , 2013, Molecular microbiology.

[8]  K. Fisher,et al.  Heterologous expression, purification and cofactor reconstitution of the reductive dehalogenase PceA from Dehalobacter restrictus. , 2012, Protein Expression and Purification.

[9]  A. Neumann,et al.  Evidence for a radical mechanism of the dechlorination of chlorinated propenes mediated by the tetrachloroethene reductive dehalogenase of Sulfurospirillum muftivorans. , 2007, Environmental science & technology.

[10]  R. Sanford,et al.  Geobacter lovleyi sp. nov. Strain SZ, a Novel Metal-Reducing and Tetrachloroethene-Dechlorinating Bacterium , 2006, Applied and Environmental Microbiology.

[11]  A. Spormann,et al.  Molecular Identification of the Catabolic Vinyl Chloride Reductase from Dehalococcoides sp. Strain VS and Its Environmental Distribution , 2004, Applied and Environmental Microbiology.

[12]  W. D. de Vos,et al.  Two distinct enzyme systems are responsible for tetrachloroethene and chlorophenol reductive dehalogenation in Desulfitobacterium strain PCE1 , 2001, Archives of Microbiology.

[13]  D. Burris,et al.  Trichloroethene Reductive Dehalogenase fromDehalococcoides ethenogenes: Sequence of tceA and Substrate Range Characterization , 2000, Applied and Environmental Microbiology.

[14]  W. D. de Vos,et al.  Purification and Molecular Characterization ofortho-Chlorophenol Reductive Dehalogenase, a Key Enzyme of Halorespiration in Desulfitobacterium dehalogenans * , 1999, The Journal of Biological Chemistry.

[15]  Christof Holliger,et al.  Reductive dechlorination in the energy metabolism of anaerobic bacteria , 1998 .

[16]  R. Matthews,et al.  Structure-based perspectives on B12-dependent enzymes. , 1997, Annual review of biochemistry.

[17]  F. Guerlesquin,et al.  Structure, function and evolution of bacterial ferredoxins. , 1988, FEMS microbiology reviews.