An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation

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.

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs

“…[15] Similarly, human catechol O-MT (COMT) in cascade with V140T AtHMT catalyzed regioselective mono-allylation of 3,4dihydroxybenzaldehyde to produce 4-allyloxy-3hydroxybenzaldehyde (8) with 48 % conversion (Table S5). [16] The identities of the products were confirmed by 1 H and 13 C NMR spectroscopy (Figures S12-S17). The conversions achieved correspond to 41 and 48 SAH regeneration cycles.…”

“…Because an iodide ion is released for each molecule of SAE formed (Figure 1 b), we initially considered using our recently published ultrasensitive halide assay. [13] However, we were concerned that background signals originating from chloride in crude cell lysates would complicate the interpretation of results. Therefore, we developed a modified assay that is insensitive to chloride, but highly sensitive to iodide.…”

Novel bacterial chloromethane degraders of a living tree fern evidenced by 13C-chloromethane incubations

“…Enzymes that use or produce halides were detected using the BRENDA database available at www.brenda-enzymes.org, and adapted from [79]. This more in-depth MAG genetic content analysis was performed for six MAGs from the phyllosphere (LEAF and LEASFWASH) and…”

Die gerichtete Evolution einer Halogenid‐Methyltransferase erlaubt die biokatalytische Synthese diverser SAM‐Analoga

“…[15] Ebenso katalysierte die humane Catechol-O-MT (COMT) in einer Kaskade mit V140T-AtHMT die regioselektive Monoallylierung von 3,4-Dihydroxybenzaldehyd, um 4-Allyloxy-3-hydroxybenzaldehyd (8) bei 48 % Umsatz zu bilden (Tabelle S5). [16] Die Identität der Produkte wurde über 1 H-und 13 Wir haben gezeigt, dass das Spektrum biokatalytischer Alkylierungen rasch durch Ausnutzung der Promiskuität einer optimierten Halogenid-Methyltransferase erweitert werden kann. Die HMT aus Arabidopsis thaliana ermçglichte es uns, drei SAM-Analoga aus preiswerten und leicht verfügbaren Alkyliodiden herzustellen.…”

“…Da pro gebildetes SAE-Molekül ein Iodidion freigesetzt wird (Abbildung 1 b), erwogen wir zunächst unseren kürzlich publizierten hochsensitiven Halogenidassay. [13] Allerdings hatten wir Bedenken, dass Hintergrundsignale, die durch das in den Zellrohlysaten vorhandene Chlorid verursacht werden, die Interpretation der Ergebnisse verkomplizieren würden. Daher entwickelten wir einen modifizierten Test, der unempfindlich für Chlorid, aber hoch sensitiv für Iodid ist.…”