%0 Journal Article
%T Guidelines for Determining the Structures of Radical SAM Enzyme-Catalyzed Modifications in the Biosynthesis of RiPP Natural Products.
%A Bushin LB
%A Seyedsayamdost MR
%J Methods Enzymol
%V 606
%N 0
%D 2018
%M 30097102
%F 1.682
%R 10.1016/bs.mie.2018.04.016
%X Radical S-adenosylmethionine (RaS) enzymes catalyze some of the most fascinating transformations in Nature. With only ~100 of the >300,000 members studied to date, it is safe to assume that a plethora of new reactions and reaction mechanisms remain to be elucidated. It is by now relatively easy to spot RaS enzymes in microbial genomes. However, to determine the reactions that they carry out, detailed structural characterization of the product(s) is necessary, a process that still represents a significant roadblock in the study of RaS enzymes. We have recently combined natural products structural elucidation along with RaS enzymology to provide a proof of concept for how the confluence of these approaches can lead to the discovery of new natural products and RaS enzyme-mediated transformations. Herein, we provide guidelines for expressing, purifying, and reconstituting a subclass of RaS enzymes that contain a so-called SPASM domain, as well as characterizing the reactions that they catalyze using a combination of HR/MSn and NMR investigations. Application of these approaches will aid in expanding the chemical and biosynthetic repertoire of RaS enzymes in the future.