The PCP degradation pathway in S. chlorophenolicum
has been extensively studied with the enzymes and genes identified and characterized (3
). One of the enzymes, PcpC, as a TeCH reductive dehalogenase, is easily damaged by H2
). PcpC-ox converts TeCH into GS-TriCH and GS-DiCH conjugates, and those conjugates cannot be further metabolized by PcpC. The pcpF
gene, next to pcpC
on the chromosome of S. chlorophenolicum
), codes for a putative GST. A BLASTP search reveals that PcpF is highly conserved in the Bacteria
domain, including enterobacteria, proteobacteria, cyanobacteria, and firmicutes (gram positive). The top 100 hits have a high degree of homology to PcpF, with the first on the list, YqjG of Bradyrhizobium
sp. strain BTAi1, displaying 63.7% identity and the last on the list, SPO3222 of Silicibacter pomeroyi
DSS-3, showing 52.6% identity by global alignments (9
). Since the homologous proteins are all hypothetical GSTs, the role of PcpF in PCP degradation is not apparent from sequence analysis. However, our biochemical and genetic analyses demonstrate that PcpF channels the GS-TriCH and GS-DiCH generated by PcpC-ox back to the PCP degradation pathway in S. chlorophenolicum
The role of PcpF-catalyzed conversion of GS-TriCH and GS-DiCH to TriCH and DiCH was demonstrated by biochemical assays with purified PcpF. Although PcpC converts TeCH to TriCH and then to DiCH without the production of the conjugates, PcpC-ox and PcpC C14S produced GS-TriCH and GS-DiCH conjugates (8
). When GS-TriCH and GS-DiCH were generated by PcpC C14S from TeCH and TriCH, PcpF converted them to TriCH and DiCH, respectively (Fig. ). The reactions catalyzed by these enzymes were proposed as shown in Fig. . When PcpF was coupled with PcpC C14S, TeCH was quantitatively converted to DiCH. Since PcpF did not use TeCH or TriCH directly, the reactions were concerted actions of PcpC C14S and PcpF.
FIG. 5. Proposed role of PcpF in the PCP degradation pathway. PcpC catalyzes two consecutive steps in PCP degradation. Both PcpC C14S and PcpC-ox catalyze the formation of GS-TriCH and GS-DiCH conjugates. PcpF channels the conjugates back to the PCP degradation (more ...)
Both PcpC and PcpF are sensitive to reactive oxygen species. It is known that the Cys-14 residue of PcpC is the target for oxidation, and PcpC-ox produced GS-TriCH and GS-DiCH (8
). PcpF has two Cys residues at positions of 53 and 248. Our site-directed mutagenesis demonstrated that Cys-53 was critical for the enzyme activity toward the conjugates, while Cys-248 was not. The Cys residues can be damaged by H2
. During PCP degradation by S. chlorophenolicum
, it is possible that only very small fractions of PcpC and PcpF are oxidatively damaged. PcpC-ox will produce very small amounts of GS-quinol conjugates, and the undamaged PcpF can convert them to TriCH and DiCH, back to the PCP degradation pathway (Fig. ). The small fraction of oxidatively damaged PcpF is not functional, which should not cause any detrimental effects to the cells.
Genetic analysis further supports the maintenance role of PcpF during PCP degradation. The presence of another carbon and energy source made a major difference for PCP degradation by the pcpF
mutant. The S. chlorophenolicum
wild type and its pcpF
disruption mutant both degraded 100 μM PCP at similar rates with glutamate, while the mutant significantly slowed down PCP degradation in the absence of glutamate. Since the cells were growing on glutamate when induced with PCP, glutamate was the major carbon and energy source for the cells. After harvested and resuspended in fresh medium without glutamate, the cells lost the major carbon and energy source. It is known that carbon-starved cells are under significant oxidative stress (11
). Thus, it is likely that glutamate-starved cells contain more PcpC-ox molecules and consequently more GS-TriCH and GS-DiCH conjugates. For the wild type, PcpF returns them back to the PCP degradation pathway. For the pcpF
mutant, the conjugates may accumulate inside the cells. The quinol moieties in the conjugates can undergo oxidation and reduction to generate more reactive oxygen species, toxic to the cells (2
). The toxic effects of conjugates to the cells were further tested with plate assays containing various concentrations of PCP. The PCP degradation assay in liquid culture was a short-time exposure to PCP (Fig. ), while the plate assay was a long-time exposure to PCP. Only small amounts of cells were spotted onto the agar surface, and the cell numbers were significantly reduced in higher dilutions. The few cells on the agar plate grew on glutamate and PCP simultaneously, and PCP could not be rapidly consumed due to low cell density. Even if GS-TriCH and GS-DiCH conjugates were produced at a very slow rate by a small fraction of PcpC-ox, they might be accumulated in the pcpF
mutant cells. The accumulation of the conjugates was suggested as the pcpF
mutant degraded PCP more slowly (Fig. ) and was more sensitive to PCP (Fig. ).
PcpF is a GST, as indicated by its ability to catalyze GSH-dependent GS-hydroquinone lyase activity and by its sequence similarity with other GSTs. Another GST, LigG, has been shown to catalyze a similar lyase reaction, removing the GS moiety from α-GS-β-hydroxypropiovanillone (7
). PcpF and LigG share only 20.1% sequence identity by global alignment (9
). The Cys-15-Pro-16 sequence at the N terminus of LigG is aligned with the Cys-53-Pro-54 sequence at the N terminus of PcpF. The same sequence has been reported to play a catalytic role in a human omega class GST, GSTO1-1, with Cys-32 forming a disulfide bond with GSH and Pro-33 stabilizing the Cys thiolate (1
). LigG and GSTO1-1 share 22.9% sequence identity, and PcpF and GSTO1-1 have 20.1% sequence identity. Although the sequence homology is not great among the three GSTs, both PcpF and LigG are likely to form disulfide bonds with the GS moiety from the conjugate substrates before the Cys-SH is regenerated with a GSH by a thiol-disulfide exchange reaction.