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Journal of Bacteriology, March 2008, p. 1615-1619, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01697-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134{triangledown}

Sara Mae Belchik and Luying Xun*

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234

Received 22 October 2007/ Accepted 20 December 2007

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 (FADH2)-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 FADH2, 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 (TcpXH) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpXH had no activity toward NADPH or riboflavin. Coupling of TcpXH and TcpA demonstrated that TcpXH provided FADH2 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 TcpXH 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.

* Corresponding author. Mailing address: Washington State University, 301 Abelson Hall, Pullman, WA 99164-4234. Phone: (509) 335-2787. Fax: (509) 335-1907. E-mail: xun{at}mail.wsu.edu

{triangledown} Published ahead of print on 28 December 2007.

Journal of Bacteriology, March 2008, p. 1615-1619, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01697-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zhang, J.-J., Liu, H., Xiao, Y., Zhang, X.-E., Zhou, N.-Y. (2009). Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3. J. Bacteriol. 191: 2703-2710 [Abstract] [Full Text]  


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