Characterization of tetrachlorohydroquinone reductive dehalogenase from Sphingomonas sp. UG30
This thesis is an investigation of the tetrachlorohydroquinone reductive dehalogenase (PcpC) from Sphingomonas sp. UG30. PcpC is the second of five enzymes that catalyze the initial degradation of pentachlorophenol in several bacterial strains. The characterization of the wildtype UG30 PcpC revealed a 94% primary sequence identity with the well-studied PcpC from Sphingobium chlorophenolicum ATCC 39723. The UG30 PcpC had temperature and pH optima of 50°C and 8.7, respectively. Reduced glutathione and a reducing agent (dithiothreitol) were necessary for activity. The S. chlorophenolicum PcpC is susceptible to severe inhibition by its substrate tetrachlorohydroquinone, necessitating the use of a mutant enzyme for kinetic studies. In contrast, the UG30 PcpC is not inhibited by tetrachlorohydroquinone, allowing kinetic studies of the wildtype UG30 PcpC. These studies have indicated an enhanced ability to dehalogenate tetrachlorohydroquinone to dichlorohydroquinone relative to the S. chlorophenolicum PcpC.;An examination of N-terminal Tyr, Ser, and Cys residues by site-directed mutagenesis indicated that Ser12 and Cys14 are essential for PcpC activity, while Tyr7 and Tyr9 do not appreciably affect activity. Ser15 was also involved in the activity of PcpC, though its role appears to be more structural in nature. An interesting observation of the Cys14Ala mutant of the UG30 PcpC was a reduced reaction rate, while the dehalogenation of tetrachlorohydroquinone to dichlorohydroquinone proceeded as in the wildtype enzyme. In contrast, the S. chlorophenolicum PcpC Cys14 mutant was found to have no change in reaction rate relative to the wildtype enzyme, however, only glutathione conjugated dead-end metabolites were formed. Thus, the role of the N-terminal Cys residue may reflect different reaction mechanisms in these two PcpC enzymes.;Chemical modification and site-directed mutagenesis of His and Lys residues in the UG30 PcpC suggested that His154 and Lys114 are involved in the orientation of glutathione in the active site of PcpC. Models of the PcpCs were also generated using the 3D-PSSM fold recognition server.;This research has elucidated important aspects of the structure-function of the UG30 PcpC as a member of the glutathione transferases and as a reductive dehalogenase.