title: Gene cloning and molecular characterization of a two-enzyme system catalyzing the oxidative detoxification of beta-endosulfan.
abstract: The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent beta-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of beta-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of beta-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either alpha-endosulfan or the metabolite of endosulfan, endosulfan sulfate.
authors.affiliation: CSIRO Entomology, Canberra, ACT 2601, Australia. Tara.Sutherland@csiro.au
authors.forename: Tara D
authors.initials: TD
authors.lastname: Sutherland
authors.forename: Irene
authors.initials: I
authors.lastname: Horne
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Russell
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004726:Endosulfan / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006843:Hydrocarbons, Chlorinated; D007306:Insecticides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D010084:Oxidation-Reduction
chemical_list: D006843:Hydrocarbons, Chlorinated; D007306:Insecticides; D005182:Flavin-Adenine Dinucleotide; D004726:Endosulfan