Biocatalytic alkylations are important reactions to obtain chemo-, regio-and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

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.

Biokatalytische Alkylierungen sind wichtige Reaktionen, um chemo-, regio-und stereoselektiv alkylierte Verbindungen zu erhalten. Dies kann mit S-Adenosyl-l-methionin (SAM)-abhängigen Methyltransferasen und SAM-Analoga erreicht werden. Kürzlich wurde gezeigt, dass eine Halogenid-Methyltransferase (HMT) aus Chloracidobacterium thermophilum SAM ausgehend von SAH und Methyliodid herstellen kann. Wir entwickelten einen Iodid-basierten Assay für die gerichtete Evolution einer HMT aus Arabidopsis thaliana und nutzten diesen, um eine V140T-Variante zu identifizieren, die auch Ethyl-, Propyl-, und Allyliodid akzeptiert, um die entsprechenden SAM-Analoga herzustellen (90, 50 und 70 % Umsatz ausgehend von 15 mg SAH). Die V140T-AtHMT wurde in einer Eintopfkaskade mit O-Methyltransferasen (IeOMT und COMT) eingesetzt, um die regioselektive Alkylierung von Luteolin und 3,4-Dihydroxybenzaldehyd zu erzielen. Obwohl eine Kaskade zur Propylierung von 3,4-Dihydroxybenzaldehyd niedrige Umsätze ergab, konnte das Propyl-SAH-Intermediat durch NMR-Spektroskopie bestätigt werden.

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