Heeb_2018_Chemosphere_207_118

LinB is a haloalkane dehalogenase found in Sphingobium indicum B90A, an aerobic bacterium isolated from contaminated soils of hexachlorocyclohexane (HCH) dumpsites. We showed that this enzyme also converts hexabromocyclododecanes (HBCDs). Here we give new insights in the kinetics and stereochemistry of the enzymatic transformation of delta-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P1delta, P2delta) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T1delta, T2delta). Enzymatic transformations of delta-HBCD, alpha1-PBCDol, one of the transformation products, and alpha2-PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters KM, vmax, kcat/KM indicated that delta-HBCD is the best LinB substrate followed by alpha2- and alpha1-PBCDol. The stereochemistry of these transformations was modeled in silico, investigating respective enzyme-substrate (ES) and enzyme-product (EP) complexes. One of the four predicted ES-complexes led to the PBCDol product P1delta, identical to alpha2-PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An SN2-like substitution of bromine at C6 of delta-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to alpha2-PBCDol. Modeling results further indicate that backside attacks at C1, C9 and C10 are reasonable too, selectively binding leaving bromide ions in a halide pocket found in LinB. Docking with alpha2-PBCDol, also allowed productive enzyme binding. A TBCD-1,5-diol with the 1S,2S,5R,6R,9S,10R-configuration is the predicted second-generation product T1delta. In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of delta-HBCD to specific PBCDols and TBCDdiols at A-resolution and predictions of their stereochemistry.