Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

Abstract

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s(-1)) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Keywords: biodegradation; channeling; molecular evolution; quinone reductase.

Fig. 1.

The initial steps in the pathway for degradation of PCP in S. chlorophenolicum via the intermediates TCBQ and TCHQ are highlighted in green. Hydroxylation of TCP by PcpB forms TCHQ directly.

Fig. 2.

TCBQ formed by PcpB forms covalent adducts with PcpB. (A) ¹⁴C incorporated into PcpB after incubation with U-¹⁴C-PCP and cofactors and PcpD as indicated for 10 min at room temperature. (B) SDS/PAGE analysis of reaction mixtures in which PcpB was incubated with PCP (100 µM) in the absence (lane 1) or presence (lane 2) of NADPH and NADH and in the presence of NADPH, NADH, and PcpD (10 μM) (lane 3) for 10 min at room temperature in 50 mM potassium phosphate, pH 7. Lane 4, molecular mass markers.

Fig. 3.

Phenotypic differences between wild-type and ΔpcpD S. chlorophenolicum. (A) Wild-type or ΔpcpD cells were spotted on ATCC 1687 agar plates containing glutamate as a carbon source. The plates were supplemented with 100 μM PCP, TCP, or TriCP and incubated in the dark for 5–7 d. (B) Products derived from U-¹⁴C-PCP are excreted into the medium by cells lacking PcpD (red), but not by wild-type cells (blue). Compounds eluting from an HPLC column were monitored by scintillation counting (Left) or by UV-Vis spectroscopy (Right).

Fig. 4.

Reduction of TCBQ by NADH in the presence and absence of PcpD. TCBQ was mixed with NADH and varying concentrations of PcpD in a stopped-flow spectrophotometer to yield final concentrations of 30 µM TCBQ, 200 µM NADH, and 0 nM (black), 200 nM (blue), 400 nM (red), or 600 nM (green) PcpD. Depletion of TCBQ was monitored at 290 nm and was fit to a single exponential (shown in black for each curve). The Inset uses a compilation of additional experimental conditions and replicates to demonstrate the dependence of the observed rates on enzyme concentration.

Fig. 5.

Products formed from PCP by PcpB in the presence and absence of PcpD. (A) The rates of reaction of TCBQ with β−ME and PcpD under the experimental conditions. (B) The amount of PCP consumed is shown in light gray bars, the amount of TCHQ formed is shown in dark gray bars, and the amount of THTH formed is shown in black bars. Reactions contained 2.8 µM PcpB, 100 µM PCP, 50 µM NADPH, 50 µM NADH, 6 mM β-ME in the presence or absence of 10 µM PcpD. The discrepancies between the amounts of PCP used and the amounts of TCHQ or THTH formed are due to (i) the rapid reaction of TCBQ with thiols, especially in the absence of PcpD; and (ii) the difficulty in monitoring relatively small amounts of PCP disappearance by HPLC, which leads to relatively large errors in quantitation.