Metabolism of Carbaryl via 1,2-Dihydroxynaphthalene by Soil Isolates Pseudomonas sp. Strains C4, C5, and C6

Abstract

Pseudomonas sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O 2 uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl 3 1-naphthol 3 1,2-dihydroxynaphthalene 3 salicylaldehyde 3 salicylate 3 gentisate 3 maleylpyruvate.Carbamate insecticides, such as carbaryl (1-naphthyl-Nmethylcarbamate), are highly toxic, have a wide range of activity, and comprise a major portion of pesticides used in the agriculture industry. Widespread and repeated use leads to pollution of soil and groundwater (5). The ester bond between N-methylcarbamic acid and 1-naphthol is responsible for carbaryl toxicity. Carbamates are competitive inhibitors of neuronal nicotinic acetylcholine receptors and acetylcholinesterase (28). N-Nitrosocarbamates and the 1-naphthol that they generate are potent mutagens and are more toxic and recalcitrant than carbaryl itself (8,22,26,27,32). Carbamate pesticides generally do not persist in the environment for a long time. In aqueous solutions, carbaryl hydrolyzes to 1-naphthol, methylamine, and CO 2 (30). Bacteria capable of degrading carbamate pesticides have been isolated from the soil (3,6,7,13,16,25). The first step in degradation is the hydrolysis of carbaryl to 1-naphthol by carbaryl hydrolase, which has been purified and characterized from various organisms (3,5,10,11,20,24). Depending on the strain, 1-naphthol is metabolized via salicylate to either gentisate or catechol (4,7,9,16,17). It has been proposed that prior to ring cleavage, 1-naphthol is hydroxylated either to 4-hydroxy-1-tetralone (1), 3,4-dihydro-dihydroxy-1(2H)-naphthalenone (31), or 1,4-naphthoquinone (25). Though 1-naphthol, salicylate, and gentisate are well-established intermediates in carbaryl degradation, the steps and enzymes responsible for the conversion of 1-naphthol to salicylate have not been demonstrated so far.In this study, we report isolation of three soil bacterium Pseudomonas sp. strains, C4, C5, and C6, utilizing carbaryl as the carbon source via 1-naphthol, salicylate, and gentisate. The aim of this study is to investigate the catabolic pathway and its regulation, with emphasis on the enzymes involved in the conversion of 1-naphthol to ...

Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4

“…1 (36). The involvement of 1,2-dihydroxynaphthalene as a metabolic intermediate was established by conducting a series of metabolic studies and demonstrating 1-naphthol-2-hydroxylase (1-NH) and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbarylgrown cells (36). The enzyme 1-NH, responsible for the conversion of 1-naphthol to 1,2-dihydroxynaphthalene, was found to be inducible, requires NAD(P)H plus flavin adenine dinucleotide (FAD) for its activity, and has not been purified or characterized to date.…”

“…One such recalcitrant compound is 1-naphthol, a high-volume industrial product widely used in the production of synthetic dyes, perfumes, and pesticides such as carbaryl (Sevin) (8,14). It is also released by microbes into the environment as a metabolic intermediate of various polycyclic aromatic compounds, including carbaryl (6,9,32,36).…”

“…strain C4, isolated in our laboratory from soil by an enrichment culture technique, utilizes carbaryl as the sole source of carbon and energy via 1-naphthol and 1,2-dihydroxynaphthalene, as shown in Fig. 1 (36). The involvement of 1,2-dihydroxynaphthalene as a metabolic intermediate was established by conducting a series of metabolic studies and demonstrating 1-naphthol-2-hydroxylase (1-NH) and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbarylgrown cells (36).…”

“…Pseudomonas sp. strain C4 was grown in 150 ml minimal salt medium in 500-ml baffled Erlenmeyer flasks at 30°C on a rotary shaker at 200 rpm (36). The medium was supplemented aseptically with carbaryl (0.1%) as the sole source of carbon.…”

“…The reaction was terminated by adjusting the pH to 2 with HCl (2 N), and the products were extracted in ethyl acetate, concentrated, and dried over anhydrous sodium sulfate. The reaction products were resolved by thin-layer chromatography (TLC) as described earlier (36) and identified by comparing R f and UV fluorescence properties with those of authentic 1-naphthol and 1,2-dihydroxynaphthalene.…”