Determination of the catalytic product of pentachlorophenol 4-monooxygenase (PcpB) and crystallization of tetrachlorohydroquinone reductive dehalogenase, dichlorohydroquinone dioxygenase and PcpB in the pentachlorophenol biodegradation pathway

Pentachlorophenol (PCP) is a priority environmental pollutant in North America that was widely used as a wide-spectrum biocide in the last century. Pentachlorophenol 4-monooxygenase (PcpB) is the first and rate-limiting enzyme in the PCP biodegradation pathway in Sphingobium chlorophenolicum ATCC 39723. Pentachlorophenol 4-monooxygenase (PcpB) catalyzes the hydroxylation of pentachlorophenol in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum. Previous studies from two different research groups proposed oppositely that the catalytic product of PcpB was tetrachlorohydroquinone (TCHQ) and tetrachlorobenzoquinone (TCBQ). We re-examined the identity of the catalytic product of PcpB, because TCHQ and TCBQ are present in a redox-equilibrium in aqueous solutions and the chemical reagents NADPH, ethyl acetate and glutathione used for the product detection in the previous studies may shift the redox-equilibrium. In this study, we investigated the effects of NADPH, ethyl acetate and glutathione on the redox-equilibrium and product distribution. Under newly designed experimental conditions, we confirmed unambiguously that the catalytic product of PcpB is TCHQ instead of TCBQ. We also propose that TCBQ may be produced non-specifically by peroxidases within the bacterial cells and that TCBQ reductase (PcpD) might act as a self-protective rather than a PCP-degradation enzyme. There is no glutathione S-transferase (GST) activity for wild type of PcpC, PcpC (C13S) mutant and a surface loop deletion mutant PcpC (LD). In the crystallization studies, PcpC (C13S), PcpB and PcpA were over-expressed and purified to >95% purity using Ni-NTA based affinity chromatography. Initial crystallization conditions have been obtained for all three enzymes from both in-house screening using the screening kits from Qiagen-Nextal and high throughput screening at the Hauptman-Woodward Institute, Buffalo, New York. Further structural determination studies will be undertaken upon obtaining large crystals suitable for X-ray diffraction data collection