Abstract
The terebellid polychaete Amphitrite ornata produces no detectable volatile halogenated secondary metabolites, but frequently inhabits coastal marine sediments heavily contaminated with anthropogenic or biogenic haloaromatic compounds. This animal contains high levels of two very unusual enzymes, dehalogenating peroxidases. We have purified and partially characterized one of these dehaloperoxidases, DHP I. DHP I is a heme enzyme (Mr = 30,790) composed of two identical subunits (Mr = 15,529) and is very rich in the amino acids aspartic acid (+ asparagine) and glutamic acid (+ glutamine). The enzyme converts trihalogenated phenols, such as 2,4,6-tribromophenol, into dihalogenated quinones. The optimum pH for this reaction is 5.0. DHP I is also active against di- and monohalogenated phenols and will oxidize bromo-, chloro-, and fluorophenols. We have identified similar dehaloperoxidase activities in other infaunal polychaetes, including halometabolite-producing species.
Highlights
Contamination of coastal marine sediments by anthropogenic haloaromatic compounds found in agricultural, industrial, and urban runoff is well known [1, 2]
We examined A. ornata to determine the basis for its tolerance of haloaromatic compounds and found that, instead of a typical oxygenase, it produces high levels of two dehalogenating peroxidases
Bromophenols and related compounds are respiratory inhibitors and presumably express their toxicity through inhibition of mitochondrial function. The toxicity of these compounds to vertebrates is well established (16 –19), but has not been as thoroughly examined in invertebrates. It is clear from field and laboratory studies that sediment contamination with bromoaromatics inhibits recruitment of non-bromometabolite-producing invertebrate species [15] and appears to select for specific organisms which may be resistant to the toxic effects of haloaromatic compounds
Summary
Materials—A. ornata was collected at low tide from mixed oyster rubble and sandy mud at Debidue flats, in the North Inlet estuary (Georgetown, SC, 33°20ЈN, 79°10ЈW) and immediately frozen on dry ice. Dehaloperoxidase Activity, Products, and Protein Assays—A. ornata dehaloperoxidase I activity was assayed on the basis of disappearance of substrate, typically 2,4,6-tribromophenol. Activity was assayed using a reaction mixture containing 50 mM KH2PO4, pH 5.0, 5 mM H2O2, from 1 to 20 M halophenol substrate, and 0.15 g of pure DHP I in a 1-ml reaction volume, for 5 min at room temperature. An internal standard (2,6-dichlorophenol) was added, and the reaction was stopped immediately by extraction with high performance liquid chromatography grade pentane. Optimum pH for enzyme activity was determined using reaction mixtures buffered to different pH values with 100 mM sodium acetate buffer (pH 3.8 – 6.0) or 50 mM potassium phosphate buffer (pH 5.0 –7.0). One unit of dehaloperoxidase activity was defined as 1 nmol of 2,4,6-tribromophenol debrominated per min.
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