Functional Expression and Characterization of Tetrachloroethene Dehalogenase From Geobacter sp

Frontiers in Microbiology
Ryuki NakamuraMasafumi Yohda

Abstract

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.

References

Apr 1, 1988·FEMS Microbiology Reviews·M Bruschi, F Guerlesquin
Jan 1, 1997·Annual Review of Biochemistry·M L Ludwig, R G Matthews
Apr 7, 2006·Applied and Environmental Microbiology·Youlboong SungFrank E Löffler
May 13, 2014·Applied and Environmental Microbiology·Anita Mac NellyTorsten Schubert
Sep 28, 2015·Trends in Biotechnology·Bat-Erdene JugderChristopher P Marquis
Mar 15, 2016·Frontiers in Microbiology·Bat-Erdene JugderMichael Manefield

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Datasets Mentioned

BETA
LC342077

Methods Mentioned

BETA
PCR
Assay
atomic absorption spectrometry
size exclusion chromatography
electron paramagnetic resonance

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