Thiomuracin is a nanomolar potency thiopeptide antibiotic with activity towards many gram-positive drug-resistant bacteria. It belongs to the family of ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products and is biosynthesized from a ribosomal precursor peptide, TbtA, by a complex array of posttranslational modifications in Thermobispora bispora (tbt). One of the several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp2 carbon. We have reconstituted the activity of TbtI in-vitro, the responsible radical S-adenosyl-methionine (radical SAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies have demonstrated that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. We have found that the enzyme functions in a mostly leader peptide independent manner. Our studies have uncovered the minimal substrate for TbtI as well as the basis for it's regioselectively that leads to the modification at a single thiazole, although six appear in the native substrate. A combination of biochemical and biophysical approaches have demonstrated that TbtI carries out catalysis unlike any previously characterized radical SAM methyltransferases. The in vitro reconstitution of TbtI, a class C radical SAM methyltransferase with an unique mechanism, further adds to the chemical versatility of radical SAM enzymes, and informs on the complexity of thiomuracin biosynthesis. These studies will facilitate generation of novel analogs of thiomuracin with desirable pharmacological properties.

Studies on the radical S-adenosylmethionine (SAM) thiazole C-methyltransferase involved in thiomuracin biosynthesis

254th National Meeting (2017)

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