Gene cloning and molecular characterization of a two-enzyme system catalyzing the oxidative detoxification of beta-endosulfan

Appl Environ Microbiol. 2002 Dec;68(12):6237-45. doi: 10.1128/AEM.68.12.6237-6245.2002.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalysis
  • Cloning, Molecular
  • Endosulfan / metabolism*
  • Flavin-Adenine Dinucleotide / metabolism
  • Hydrocarbons, Chlorinated*
  • Insecticides / metabolism*
  • Molecular Sequence Data
  • Mycobacterium / genetics
  • Mycobacterium / metabolism*
  • Open Reading Frames
  • Oxidation-Reduction


  • Hydrocarbons, Chlorinated
  • Insecticides
  • Flavin-Adenine Dinucleotide
  • Endosulfan