Flavin-adenine dinucleotide (FAD)-dependent halogenases are widespread in natural product biosynthetic gene clusters and have been demonstrated to employ small organic molecules as substrates for halogenation, as well as substrates that are tethered to carrier proteins (CPs). Despite numerous reports of FAD-dependent halogenases utilizing CP-tethered substrates, only a few have been biochemically characterized due to limited accessibility to the physiological substrates. Here, we describe a method for the preparation of acyl-S-CP substrates and their use in biochemical assays to query the activity of FAD-dependent halogenases. Furthermore, we describe a mass spectrometry-based method for the characterization of acyl-S-CP substrates and the corresponding halogenated products generated by the halogenases. Finally, we test the substrate specificity of a physiological chlorinase and a physiological brominase from marine bacteria, and, for the first time, demonstrate the distinct halide specificity of halogenases. The methodology described here will enable characterization of new halogenases employing CP-tethered substrates.
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