Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134.
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 (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 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 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.
Belchik
Sara Mae
SM
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234, USA.
Xun
Luying
L
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism; D002733:Chlorophenols / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D038181:FMN Reductase / Q000235:genetics / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D013696:Temperature
D001426:Bacterial Proteins; D016227:Benzoquinones; D002733:Chlorophenols; D005182:Flavin-Adenine Dinucleotide; C004532:quinone; D005486:Flavin Mononucleotide; D038181:FMN Reductase; D016660:NAD(P)H Dehydrogenase (Quinone); C024564:2,4,6-trichlorophenol