Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum

Abstract

Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The k(cat) for PCP (0.024 s(-1)) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure-activity studies reveal that substrate binding and activity are enhanced by a low pK(a) for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H(2)O(2) in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 to 100% with different substrates. The extent of uncoupling is increased by the presence of bulky substituents at position 3, 4, or 5 and decreased by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with tetrachlorohydroquinone (TCHQ), a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ.

Figure 1

Pathway for degradation of PCP in Sphingobium chlorophenolicum. PcpB, PCP hydroxylase; PcpD, TCBQ reductase; PcpC, TCHQ dehalogenase; PcpA, 2,6-dichlorohydroquinone dioxygenase; GSH, glutathione; HGT, horizontal gene transfer.

Figure 2

Dependence of PCP hydroxylase activity on FAD. Comparison of activities of PCP hydroxylase samples differentially loaded with FAD. Reaction mixtures containing 5 μM PCP hydroxylase, 100 μM PCP, 6 mM β-ME, and 300 μM NADPH were incubated for 3 min at room temperature and THTH was quantified by HPLC.

Figure 3

Representative HPLC traces showing the products formed by PCP hydroxylase from PCP, TCP, and TCHQ. Each substrate (100 μM) was incubated in 50 mM potassium phosphate, pH 7.5, containing 6 mM β-ME and 25 μM ascorbate, for 1 min at room temperature. PCP hydroxylase (10 μM) and NADPH (300 μM) were added as indicated. Since the various compounds absorb optimally at different wavelengths, signals detected at different wavelengths are overlaid in the chromatograms. Green, 295 nm; blue, 310 nm; red, 350 nm. p-Nitrophenol was used as an internal standard.

Figure 4

Relationship between reaction velocity and substrate concentration for A) PCP; B) TCP; C) 2,6-DCP; and D) TCHQ. Reactions were carried out in 50 mM potassium phosphate, pH 7.0, containing 160 μM NADPH, 6 mM β-ME, 25 μM ascorbate, and PCP hydroxylase.

Figure 5

Overlay of representative difference spectra for the flavin of PCP hydroxylase (7.1 μM) in the presence vs. absence of various ligands: PCP (red, 15 μM), TCP (black, 18 μM), 2,4,6-TriCP (blue, 80 μM) and 3,5-DCP (green, 2500 μM).

Figure 6

Perturbation of the flavin spectrum at 388 nm by binding of PCP (A), TCP (B) and 2,6-DCP (C).

Figure 7

Summary of binding constants, pK_as and logP values for various chlorinated phenols.

Figure 8

Stereo view of the active site of p-hydroxybenzoate hydroxylase showing the substrate (p-hydroxybenzoate) in grey and a C4a-hydroperoxyflavin modeled in place of the FAD in the “in” position (PDB 1PBE).

Scheme 1

Scheme 2