New Metabolites in the Degradation of α- and γ-Hexachlorocyclohexane (HCH): Pentachlorocyclohexenes Are Hydroxylated to Cyclohexenols and Cyclohexenediols by the Haloalkane Dehalogenase LinB from Sphingobium indicum B90A

Abstract

Technical hexachlorocyclohexane (HCH) and lindane are obsolete pesticides whose former production and use led to widespread contaminations posing serious and lasting health and environmental risks. Out of nine possible stereoisomers, alpha-, beta-, gamma-, and delta-HCH are usually present at contaminated sites, and research for a better understanding of their biodegradation has become essential for the development of appropriate remediation technologies. Because haloalkane dehalogenase LinB was recently found responsible for the hydroxylation of beta-HCH, delta-HCH, and delta-pentachlorocyclohexene (delta-PCCH), we decided to examine whether beta- and gamma-PCCH, which can be formed by LinA from alpha- and gamma-HCH, respectively, were also converted by LinB. Incubation of such substrates with Escherichia coli BL21 expressing functional LinB originating from Sphingobium indicum B90A showed that both beta-PCCH and gamma-PCCH were direct substrates of LinB. Furthermore, we identified the main metabolites as 3,4,5,6-tetrachloro-2-cyclohexene-1-ols and 2,5,6-trichloro-2-cyclohexene-1,4-diols by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. In contrast to alpha-HCH, gamma-HCH was not a substrate for LinB. On the basis of our data, we propose a modified gamma-HCH degradation pathway in which gamma-PCCH is converted to 2,5-cyclohexadiene-1,4-diol via 3,4,5,6-tetrachloro-2-cyclohexene-1-ol and 2,5,6-trichloro-2-cyclohexene-1,4-diol.