Swiss Federal Institute for Materials Science and Technology (Empa), Laboratory of Advanced Analytical Technologies, Überlandstrasse 129, CH-8600, Dübendorf, Switzerland.
Swiss Federal Institute for Materials Science and Technology (Empa), Laboratory of Advanced Analytical Technologies, Überlandstrasse 129, CH-8600, Dübendorf, Switzerland; Zurich University of Applied Sciences, Institute of Chemistry and Biological Chemistry, Reidbach, 8820 Wädenswil, Switzerland.
Chemosphere. 2018 Sep;207:118-129. doi: 10.1016/j.chemosphere.2018.05.057. Epub 2018 May 12.
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 δ-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P, P) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T, T). Enzymatic transformations of δ-HBCD, α-PBCDol, one of the transformation products, and α-PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters K, v, k/K indicated that δ-HBCD is the best LinB substrate followed by α- and α-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 P, identical to α-PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An S2-like substitution of bromine at C6 of δ-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to α-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 α-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 T. In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of δ-HBCD to specific PBCDols and TBCDdiols at Å-resolution and predictions of their stereochemistry.
林 B 是一种卤代烷脱卤酶，存在于从六氯环己烷（HCH）倾倒场污染土壤中分离出的需氧细菌 Sphingobium indicum B90A 中。我们表明，该酶还可以转化六溴环十二烷（HBCDs）。在这里，我们提供了关于 δ-HBCD 酶促转化的动力学和立体化学的新见解，该转化导致两种五溴环十二烷醇（PBCDols）作为第一代（P，P）和两种四溴环十二烷二醇（TBCDdiols）作为第二代产物（T，T）的形成。研究并通过 Michaelis-Menten（MM）动力学对 δ-HBCD、转化产物之一的 α-PBCDol 及其对映体 α-PBCDol 的酶促转化进行了建模。相应的 MM 参数 K、v、k/K 表明，δ-HBCD 是 LinB 的最佳底物，其次是 α-PBCDol 和 α-PBCDol。通过计算机模拟对这些转化的立体化学进行了建模，研究了各自的酶-底物（ES）和酶-产物（EP）复合物。四个预测的 ES 复合物之一导致 PBCDol 产物 P 的形成，与 1R，2R，5S，6R，9R，10S-构型的 α-PBCDol 相同。LinB 的 Asp-108 取代 δ-HBCD 上的 C6 溴原子，并随后水解烷基-酶，导致形成 α-PBCDol。建模结果进一步表明，C1、C9 和 C10 的背面攻击也是合理的，选择性地将离去的溴离子结合在 LinB 中发现的卤化物口袋中。与 α-PBCDol 对接也允许有效的酶结合。具有 1S，2S，5R，6R，9S，10R-构型的 TBCD-1,5-二醇是预测的第二代产物 T。总之，体外和计算机模拟的结果现在允许在 Å 分辨率下详细描述 δ-HBCD 的逐步酶脱卤羟基化反应，以及对特定的 PBCDols 和 TBCDdiols 的立体化学预测。
