Journal article
Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134
Journal of bacteriology, Vol.190(5), pp.1615-1619
03/2008
Handle:
https://hdl.handle.net/2376/107070
PMCID: PMC2258691
PMID: 18165297
Abstract
The
tcpRXABCYD
operon of
Cupriavidus necator
JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH
2
)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH
2
, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (
tcpX
and
tcpB
) were cloned, overexpressed, and purified in
Escherichia coli
. TcpX was purified as a C-terminal His tag fusion (TcpX
H
) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX
H
had no activity toward NADPH or riboflavin. Coupling of TcpX
H
and TcpA demonstrated that TcpX
H
provided FADH
2
for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX
H
in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.
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Details
- Title
- Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134
- Creators
- Sara Mae Belchik - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234Luying Xun - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234
- Publication Details
- Journal of bacteriology, Vol.190(5), pp.1615-1619
- Academic Unit
- Molecular Biosciences, School of
- Publisher
- American Society for Microbiology (ASM)
- Identifiers
- 99900546992801842
- Language
- English
- Resource Type
- Journal article