Exploring the mechanism of action of lysine 5,6-aminomutase using EPR and ENDOR spectroscopies.

Radical enzymes orchestrate challenging chemical transformations by devising strategies to tame the highly reactive radical intermediates. Electron paramagnetic resonance (EPR) spectroscopy is the most suitable technique to study various aspects of the radical enzymes. Lysine 5,6-aminomutase (5,6-LAM) is one such radical enzyme and employs coenzyme B and pyridoxal 5'-phosphate (PLP) to catalyze the 1,2-amino shift reaction through a radical mechanism. 5,6-LAM accepts either d-lysine or l-β-lysine as the substrate. EPR and electron nuclear double resonance (ENDOR) spectroscopies have played major roles in deciphering the mechanism of action of 5,6-LAM, while density functional theoretical (DFT) computation and synthetic isotopologues have played supporting roles. This comprehensive toolkit has revealed that 5,6-LAM undergoes large-scale conformational movement to bring PLP and coenzyme B close together, which allows the reaction to progress. The conformational change also closes the active site, which protects the radical intermediates and enables their transformation to product without unwanted side reactions. The substrate-related radical (S), which is spin-coupled with Co generated from homolysis of the CoC bond in coenzyme B, was unequivocally characterized when a substrate analog, 4-thia-l-lysine, and isotopologues of it were reacted with 5,6-LAM. Studies with substrate analogs revealed a unique "odd-even" correlation with opening of the closed state. Moreover, mutagenesis studies identified the contributions that conserved residues in 5,6-LAM make toward binding of the substrate. Further studies with a cofactor analog, PLP-N-oxide, have shed light on various aspects of the mechanism of action of 5,6-LAM.