In the loop: Engineering of the surface loop in haloalkane dehalogenases affects their enantiodiscrimination behavior. The temperature dependence of the enantioselectivity (lnE versus 1/T) of β‐bromoalkanes by haloalkane dehalogenases is reversed (red data points) by deletion of the surface loop; the selectivity switches back when an additional single‐point mutation is made. This behavior is not observed for α‐bromoesters.

Gezielte Mutationen der Oberflächenschleife von Halogenalkan‐Dehalogenasen haben Auswirkungen auf die Enantiodiskriminierung. Die Temperaturabhängigkeit der Enantioselektivität (siehe lnE‐1/T‐Kurve) von β‐Bromalkanen kehrt sich bei einer Deletion der Oberflächenschleife um (rote Datenpunkte), was bei Einführung einer zusätzlichen Einzelpunktmutation wieder rückgängig gemacht wird; bei α‐Bromestern findet keine Änderung statt.

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␤-Hexachlorocyclohexane (␤-HCH) is the most recalcitrant among the ␣-, ␤-, ␥-, and ␦-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of ␥-HCH in Sphingomonas paucimobilis UT26, metabolizes ␤-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.The chlorinated insecticidal formation of a technical mixture of hexachlorocyclohexane (t-HCH) consisting of ␣-, ␤-, ␥-, and ␦-isomers has been used worldwide. Many countries, however, have now prohibited its use because of its toxicity and persistence in upland soil, but several contaminated sites remain throughout the world. Among these isomers, ␤-HCH is the most recalcitrant in the environment due to its chemical stability (1). Bacterial strains that degrade ␤-HCH have been reported (6,20), but the enzyme involved in the degradation process remains unknown.Sphingomonas paucimobilis UT26 utilizes ␥-HCH as a sole source of carbon and energy under aerobic conditions (3). The degradation pathway of ␥-HCH in this bacterium includes two steps of dehydrochlorination, two steps of hydrolytic dehalogenation, and one dehydrogenation step, catalyzed by LinA, LinB, and LinC, respectively, leading to the formation of 2,5-dichlorohydroquinone, which undergoes further degradation (16). The enzyme ␥-HCH dehydrochlorinase LinA mediates the metabolism of ␣-, ␥-, and ␦-HCH but not that of the ␤-isomer (11). We demonstrate here that UT26 is able to transform ␤-HCH and that the activity is derived from the haloalkane dehalogenase LinB.The activity of S. paucimobilis UT26 and its spontaneous linA deletion mutant YO5 (15) was assayed for the degradation of t-HCH (India Pesticides, Lucknow, India), which consists of ␣ (67.4%), ␤ (6.8%), ␥ (17.3%), and ␦ (7.4%)-isomers of HCH. Briefly, a small amount of a bacterial colony, grown on 1/3 Luria broth-agar medium (7) at 30°C, was picked and suspended (about 20 mg of cells/ml, final concentration) in the assay solution, 17 M t-HCH in W medium (3). After incubation at 30°C for 12 h, the assay solution was extracted with an equal volume of ethyl acetate and analyzed by a Shimadzu GC-17A gas chromatograph (GC) equipped with an electron capture 63 Ni detector (Shimadzu, Kyoto, Japan) and an Rtx-1 capillary column (30 m by 0.25 mm by 0.25 m; Restek). The column temperature was increased from 100 to 260°C at a rate of 20°C/min, and the gas flow rate was 30 ml/min. During the period of incubation, UT26 completely degraded all four HCH isomers, while YO5 degraded only ␤-HCH (data not shown). One unit of ␤-HCH degradation activity was defined as the activity required for the transformation of 1 mol of ␤-HCH per min. The activity was calculated using linear range in the first several hours of reaction. When W medium containing 17 M ␤-HCH was used as the assay medium, UT26 and YO5 cells degraded ␤-HCH linearly during the first 6 h at a rate of 1.5 ϫ 10 Ϫ6 and 1.6 ϫ 10 Ϫ6 U/mg of cells, respectively. No apparent difference i...