Mechanistic investigation into post-translational modifications catalyzed by radical S-adenosylmethionine enzymes

Abstract

Radical S-adenosyl-L-methionine (SAM) enzymes form one of the largest enzyme superfamilies and are found in all kingdoms of life. They catalyze a wide diversity of reactions but are united by the use of a [4Fe-4S] cluster and SAM to generate a 5'-dAdo radical which initiates catalysis through H-atom abstraction and formation of a substrate radical intermediate. Trapping and characterizing radical intermediates is challenging due to their high reactivity, and therefore many reactions catalyzed by radical SAM enzymes contain proposed intermediates that lack experimental evidence. This work seeks to trap and characterize radical intermediates to test proposed mechanisms of radical SAM enzymes involved in the post-translational modification of glycyl radical enzymes (GRE) and ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. This work primarily made use of electron paramagnetic resonance (EPR) spectroscopy, enzyme activity assays, and site-directed mutagenesis techniques. Mechanistic investigation of the enzyme glycerol dehydratase activating enzyme (GD-AE) showed that it produced the SAM cleavage product 5'-deoxyadenosine and not 5'-deoxy-5'- methylthioadenosine (MTA) when activating glycerol dehydratase (GD) as previously reported. Spectroscopic characterization of the splicease enzyme PcpX supported the hypothesis that it coordinates three [4Fe-4S] clusters. These results helped form mechanistic proposals for alpha-keto- beta-amino acid formation during RiPP maturation. Mechanistic investigations of the radical SAM epimerases from proteusin (OspD) and epipeptide (EpeE) RiPP natural product families identified similarities and differences in their catalytic mechanisms. Both enzymes initiate catalysis by abstraction of H alpha from a substrate Val or Ile residue to form a C alpha radical intermediate. Trapping of the C alpha radicals in both OspD and EpeE was made possible by mutating the quenching cysteine (Cys) to serine (Ser). C alpha radicals trapped in the active site of OspD or EpeE exhibited variable hyperfine coupling to H beta or the amide proton, indicating different conformations. Collectively, these studies provided key insights into radical SAM enzyme mechanism during post-translational modification of GREs and RiPPs that will aid future bioengineering efforts to produce novel peptide therapeutics.