Insights into Radical SAM Enzyme Mechanism from Lysine‐2,3‐aminomutase And An S ‐ adenosyl‐L‐methionine Analog

Radical SAM ( S ‐adenosyl‐L‐methionine) enzymes constitute one of the largest enzyme superfamilies with ~113,750 members from all domains of life. Upon conversion of substrate to product, they perform a wide variety of chemical reactions, such as isomerizations (in antibiotic synthesis), sulfur atom insertion (in vitamin synthesis), and bond cleavage (in DNA repair). When human radical SAM enzymes fail, disease states can ensue, including increased susceptibility to viral infection, congenital heart disease, retrovirus infection, and cofactor deficiencies. Prior to generating product, all radical SAM enzymes generate a highly reactive species, the 5′‐deoxyadenosyl (dAdo) radical. Despite its high reactivity, the dAdo radical does not initiate damaging uncontrolled radical proliferation, but instead consistently reacts with only one specific substrate‐based hydrogen atom within each radical SAM enzyme; we hypothesize that this precision is guided by the protein environment and that electron transfer is mediated by an iron‐sulfur metallocluster. Employing various spectroscopic techniques, this research investigates a universal radical SAM enzyme mechanism. Due to its reactivity, the dAdo radical has never been spectroscopically observed, making mechanistic inquiry difficult; to overcome this obstacle, we synthesized a SAM analog which generates a stabilized radical ‐ the anAdo radical. Because the radical on this anAdo species is distributed over three carbon atoms instead of one carbon, it reacts more slowly than the dAdo radical, allowing for visualization of mechanistic intermediate states through various spectroscopies including both Electron Paramagnetic Resonance and Electron Nuclear Double Resonance spectroscopies. Our work used this SAM analog to examine the radical SAM enzyme lysine‐2,3‐aminomutase, an enzyme that shifts an amino group from the second to third carbon on the amino acid lysine, forming an antibiotic precursor. These experiments showed that, upon breakage of the carbon‐sulfur bond in SAM to generate the dAdo radical, the now radical‐based carbon moves less than half the length of a carbon‐carbon bond – an incredibly short distance; this illustrates the tight control exerted by the protein architecture on the newly formed radical. While these observations generated the hypothesis that this tight active site control exists in all radical SAM enzymes, we continue to probe how the protein and iron‐sulfur cluster work in concert to constrain electron movement in these exquisitely designed radical SAM active sites. Support or Funding Information 5R01GM054608‐16Radical Generation in SAM and SAM analog