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The degradation of alpha and beta isomers of endosulfan by a two-member bacterial co-culture was studied. Results were similar whether the two isomers were present individually or together, as in technical endosulfan. The degradation of both isomers was accompanied by the formation of endosulfan diol and endosulfan lactone. Accumulation of the metabolite, endosulfan sulfate was, however, not observed during the reaction with either of the isomers. The microbial degradation of endosulfan isomers was also accompanied by a decrease in its toxicity to the test organism Tubifex tubifex Müller.

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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...

Edited by Wolfgang PetiSerine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-Å resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.

Hexachlorocyclohexane dehydrochlorinase (LinA) mediates dehydrochlorination of γ-HCH to 1, 3, 4, 6-tetrachloro-1,4-cyclohexadiene that constitutes first step of the aerobic degradation pathway. We report the 3.5 Å crystal structure of a thermostable LinA-type2 protein, obtained from a soil metagenome, in the hexagonal space group P6322 with unit cell parameters a = b = 162.5, c = 186.3 Å, respectively. The structure was solved by molecular replacement using the co-ordinates of LinA-type1 that exhibits mesophile-like properties. Structural comparison of LinA-type2 and -type1 proteins suggests that thermostability of LinA-type2 might partly arise due to presence of higher number of ionic interactions, along with 4% increase in the intersubunit buried surface area. Mutational analysis involving the differing residues between the -type1 and -type2 proteins, circular dichroism experiments and functional assays suggest that Q20 and G23 are determinants of stability for LinA-type2. It was earlier reported that LinA-type1 exhibits enantioselectivity for the (−) enantiomer of α-HCH. Contrastingly, we identified that -type2 protein prefers the (+) enantiomer of α-HCH. Structural analysis and molecular docking experiments suggest that changed residues K20Q, L96C and A131G, vicinal to the active site are probably responsible for the altered enantioselectivity of LinA-type2. Overall the study has identified features responsible for the thermostability and enantioselectivity of LinA-type2 that can be exploited for the design of variants for specific biotechnological applications.