Cloning and Sequencing of a 2,5-Dichlorohydroquinone Reductive Dehalogenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane by Sphingomonas paucimobilis |
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| Authors: | Keisuke Miyauchi Seug-Kyo Suh Yuji Nagata Masamichi Takagi |
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| Affiliation: | Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan,1. and Department of Environmental Management, Shinil Christian College, Susung-ku, Taegu 706-020, Korea2. |
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| Abstract: | Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that γ-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117–3125, 1994). In the present study, we cloned and characterized a gene, designated linD, directly involved in the degradation of 2,5-DCHQ. The linD gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione S-transferase family. When LinD was overproduced in Escherichia coli, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the linD gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing E. coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the linD region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of γ-HCH by S. paucimobilis UT26. |
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