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An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation
(2020)
Abstract
Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium‐dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 μM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well‐established GC‐MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide‐producing enzymes.
Promiscuous Dehalogenase Activity of the Epoxide Hydrolase CorEH from Corynebacterium sp. C12
(2021)
Haloalkane dehalogenases and epoxide hydrolases are phylogenetically related and structurally homologous enzymes that use nucleophilic aspartate residues for an SN2 attack on their substrates. Despite their mechanistic similarities, no enzymes are known that exhibit both epoxide hydrolase and dehalogenase activity. We screened a subset of epoxide hydrolases, closely related to dehalogenases, for dehalogenase activity and found that the epoxide hydrolase CorEH from Corynebacterium sp. C12 exhibits promiscuous dehalogenase activity. Compared to the hydrolysis of epoxides like cyclohexene oxide (1.41 μmol min–1 mg–1), the dehalogenation of haloalkanes like 1-bromobutane (0.25 nmol min–1 mg–1) is about 5000-fold lower. In addition to the activity with 1-bromobutane, dehalogenase activity was detected with other substrates like 1-bromohexane, 1,2-dibromoethane, 1-iodobutane, and 1-iodohexane. This study shows that dual epoxide hydrolase and dehalogenase activity can be present in one naturally occurring protein scaffold.