Identification of opdA , a Gene Involved in Biodegradation of the Endocrine Disrupter Octylphenol

Abstract

Octylphenol (OP) is an estrogenic detergent breakdown product. Structurally similar nonylphenols are transformed via type II ispo substitution, resulting in the production of hydroquinone and removal of the branched side chain. Nothing is known, however, about the gene(s) encoding this activity. We report here on our efforts to clone the gene(s) encoding OP degradation activity from Sphingomonas sp. strain PWE1, which we isolated for its ability to grow on OP. A fosmid library of PWE1 DNA yielded a single clone, aew4H12, which accumulated a brown polymerization product in the presence of OP. Sequence analysis of loss-of-function transposon mutants of aew4H12 revealed a single open reading frame, opdA, that conferred OP degradation activity. Escherichia coli subclones expressing opdA caused OP disappearance, with the concomitant production of hydroquinone and 2,4,4-trimethyl-1-pentene as well as small amounts of 2,4,4-trimethyl-2-pentanol. These metabolites are consistent with a type II ipso substitution reaction, the same mechanism described for nonylphenol biodegradation in other sphingomonads. Based on opdA's sequence homology to a unique group of putative flavin monooxygenases and the recovery of hydroxylated OP intermediates from E. coli expressing opdA, we conclude that this gene encodes the observed type II ipso substitution activity responsible for the initial step in OP biodegradation.Nonylphenol (NP) and octylphenol (OP), collectively referred to as alkylphenols, are detergent breakdown products with highly branched side chains that act as endocrine disrupters and are known to exhibit weak estrogenic activity (17). Tabira et al. (33) have shown that alkylphenols bind to recombinant human estrogen receptors in a dose-dependent fashion. Alkylphenols induce feminization in male amago salmon (26), cause changes in the sex ratio toward females and increase the frequency of intersexuality in Pacific oysters (27), and inhibit testicular growth in male rainbow trout during maturation (20).Given the potential of alkylphenols to cause harm to fish and other sensitive aquatic organisms, much interest has been focused on understanding the potential of microorganisms to degrade alkylphenols. Several organisms have been reported to degrade NP or OP when oxygen is available, including several fungal species (4, 22), psychrotrophic and psychrophilic Pseudomonas species (31), and the sewage sludge isolates Sphingobium xenophagum Bayram (15), Sphingomonas cloacae (12), Sphingomonas sp. strain TTNP3 (35), and Sphingobium amiense strain YT T (36). The degradation mechanism for specific NP isomers has been examined biochemically for both Sphingobium xenophagum Bayram and Sphingomonas strain TTNP3. Both strains metabolize various NP isomers by type II ipso substitution. Growth on NP appears to be limited to those isomers that contain fully substituted alpha carbons on the alkyl side chain (5, 14).There have been examples of ipso substitution as a mechanism for degradation of halogenated phenols by cytochrome P450s (2, 29), ...

Two identical nonylphenol monooxygenase genes linked to IS6100 and some putative insertion sequence elements in Sphingomonas sp. NP5

“…An additional spike of NP mix (0.2 mM) did not support further growth. Such low growth on NP or OP has been observed in the degradation by other degraders (Porter & Hay, 2007;Tanghe et al, 1999). This might be caused by the toxicity of the metabolites from NP mix .…”

“…PWE1 (opdA PWE1 gene product, EU002557). Porter & Hay (2007) reported that a recombinant E. coli strain harbouring opdA PWE1 was able to degrade an OP isomer, 4-(29,49,49-trimethylpentyl)phenol. More recently, Porter et al (2012) successfully cloned an opdA homologue (opdA Bayram and opdA TTNP3 ) from NP-degrading strains Bayram and TTNP3, respectively.…”

“…Furthermore, it was found that this degradation mechanism could be expanded for degradation of a structural homologue, bisphenol A, as well as most NP isomers in technical-grade NP (Gabriel et al, 2008;Kolvenbach et al, 2007). Porter & Hay (2007) reported isolation of an OP-degrading bacterium, Sphingomonas sp. PWE1, and its OP 4-monooxygenase gene (opdA).…”

Biodegradation of sulfamethoxazole and other sulfonamides by Achromobacter denitrificans PR1

“…BR1, PR1 cell-free extracts did not show significant degrading activity, irrespective of NADH presence, suggesting that different co-factors may be involved on SMX breakdown in these organisms. The hydroxylation of substituted aromatic com-pounds can be promoted by different catalysts, such as flavoprotein monooxygenases [33,34] or cytochromes P450 [35]. Therefore, further studies are needed to elucidate if the catalysts involved on sulfonamide degradation by strains PR1 and BR1 belong to one of these protein families.…”

ipso -Hydroxylation and Subsequent Fragmentation: a Novel Microbial Strategy To Eliminate Sulfonamide Antibiotics

“…Flavoprotein monooxygenases (33,34) and cytochromes P450 (25) are known to catalyze electrophilic hydroxylations of substituted aromatic compounds, whereby hydroperoxo-flavin (35) and hydroperoxo-iron (44) species, respectively, act as the donor of electrophilic oxygen. It remains to be elucidated whether the hydroxylation activity of the Microbacterium sp.…”