Dataset Information

A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.

ABSTRACT: Pentachlorophenol (PCP) hydroxylase, the first enzyme in the pathway for degradation of PCP in Sphingobium chlorophenolicum, is an unusually slow flavin-dependent monooxygenase (k(cat) = 0.02 s?¹) that converts PCP to a highly reactive product, tetrachlorobenzoquinone (TCBQ). Using stopped-flow spectroscopy, we have shown that the steps up to and including formation of TCBQ are rapid (5-30 s?¹). Before products can be released from the active site, the strongly oxidizing TCBQ abstracts an electron from a donor at the active site, possibly a cysteine residue, resulting in an off-pathway diradical state that only slowly reverts to an intermediate capable of completing the catalytic cycle. TCBQ reductase, the second enzyme in the PCP degradation pathway, rescues this nonproductive complex via two fast sequential one-electron transfers. These studies demonstrate how adoption of an ancestral catalytic strategy for conversion of a substrate with different steric and electronic properties can lead to subtle yet (literally) radical changes in enzymatic reaction mechanisms.

SUBMITTER: Rudolph J

PROVIDER: S-EPMC4204890 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.

Rudolph Johannes J Erbse Annette H AH Behlen Linda S LS Copley Shelley D SD

Biochemistry 20141006 41

Pentachlorophenol (PCP) hydroxylase, the first enzyme in the pathway for degradation of PCP in Sphingobium chlorophenolicum, is an unusually slow flavin-dependent monooxygenase (k(cat) = 0.02 s⁻¹) that converts PCP to a highly reactive product, tetrachlorobenzoquinone (TCBQ). Using stopped-flow spectroscopy, we have shown that the steps up to and including formation of TCBQ are rapid (5-30 s⁻¹). Before products can be released from the active site, the strongly oxidizing TCBQ abstracts an electr ...[more]

PMID: 25238136

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Project description:Pentachlorophenol (PCP) is a highly toxic pesticide that was first introduced in the 1930s. The alphaproteobacterium Sphingobium chlorophenolicum, which was isolated from PCP-contaminated sediment, has assembled a metabolic pathway capable of completely degrading PCP. This pathway produces four toxic intermediates, including a chlorinated benzoquinone that is a potent alkylating agent and three chlorinated hydroquinones that react with O2 to produce reactive oxygen species (ROS). RNA-seq analysis revealed that PCP causes a global stress response that resembles responses to proton motive force uncoupling and membrane disruption, while surprisingly, little of the response resembles the responses expected to be produced by the PCP degradation intermediates. Tn-seq was used to identify genes important for fitness in the presence of PCP. By comparing the genes that are important for fitness in wild-type S. chlorophenolicum and a non-PCP-degrading mutant, we identified genes that are important only when the PCP degradation intermediates are produced. These include genes encoding two enzymes that are likely to be involved in protection against ROS. In addition to these enzymes, the endogenous levels of other enzymes that protect cells from oxidative stress appear to mitigate the toxic effects of the chlorinated benzoquinone and hydroquinone metabolites of PCP. The combination of RNA-seq and Tn-seq results identify important mechanisms for defense against the toxicity of PCP. IMPORTANCE Phenolic compounds such as pentachlorophenol (PCP), triclosan, and 2,4-dichlorophenoxyacetic acid (2,4-D) represent a common class of anthropogenic biocides. Despite the novelty of these compounds, many can be degraded by microbes isolated from contaminated sites. However, degradation of this class of chemicals often generates toxic intermediates, which may contribute to their recalcitrance to biodegradation. We have addressed the stresses associated with degradation of PCP by Sphingobium chlorophenolicum by examining the transcriptional response after PCP exposure and identifying genes necessary for growth during both exposure to and degradation of PCP. This work identifies some of the mechanisms that protect cells from this toxic compound and facilitate its degradation. This information could be used to engineer strains capable of improved biodegradation of PCP or similar phenolic pollutants.

Dataset Information

A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.

Publications

A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.

Similar Datasets

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