| 2,4,6-Trichlorophenol (TCP) is a priority pollutant introduced into the environment through numerous sources including preservations of wood and leather as well as kraft pulp mill bleaching. The removal of TCP from the environment can be accomplished through bioaugmentation, a bioremediation approach involving addition of specific microorganisms to minimize the contaminant. Cupriavidus necator JMP134 is an attractive bioaugmentation agent as it is not only able to metabolize TCP but also utilizes it as a sole carbon and energy source. The degradation pathway has been elucidated and the enzymes responsible have been localized to the tcpRXABCYD gene cluster on the chromosome. While the functional roles of tcpR, tcpA, tcpC, and tcpD were previously identified in the metabolism of TCP, the roles of the tcpX, tcpB, and tcpY gene products had not been determined and were the focus of our studies. The first enzyme of the degradation, the FADH2-utilizing monooxygenase TcpA, requires reduced flavin to convert TCP to 6-chloro-p-hydroxyquinone. Sequence analysis indicated both tcpX and tcpB were potential flavin reductases that would satisfy the partner flavin reductase role. Both proteins were purified and their enzymatic activities were determined. TcpX was found to be the active NADH:flavin oxidoreductase. In coupled assays, TcpX acts as the partner flavin reductase to TcpA. Purified TcpB could not reduce flavin, instead acting to reduce quinone substrates. In coupled assays, TcpB reduces the TcpA reaction product, 6-chloro-p-hydroxyquinone, to 6-chloro-p-hydroxyquinol. This enzymatic reduction was further studied and found to minimize the detrimental side reactions of the 6-chloro-p-hydroxyquinone with cellular thiols. Computational analysis indicates TcpY is a beta-barrel outer membrane protein. Constitutive expression of tcpY increases the uptake of TCP in two C. necator species. TcpY is thus characterized as an outer membrane transporter for TCP. Our work completes the identification of the enzymes in the TCP degradative pathway for C. necator JMP134, and the mechanism for TCP entry into the cell has been determined. Our findings have the potential to be utilized in the bioengineering of bacteria for bioremediation of TCP. |
