Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme

The biosynthesis of branched alkoxy groups, such as the unique <i>t</i>-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical <i>S</i>-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on <i>p</i>-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also <i>sec</i>-butyl and <i>t</i>-butyl groups. To our knowledge, this is the first <i>in vitro</i> reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a <i>t</i>-butyl group.