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

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

^* To whom correspondence should be addressed. Email: xun{at}mail.wsu.edu.

	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 (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, over-expressed, 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 towards NADPH or riboflavin. Coupling TcpX_H and TcpA demonstrated TcpX_H 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 TcpX_H in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB can enhance TcpA activity. Further, we showed that TcpB was more effective to reduce 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.