Reaction of Pyridoxal-5′-phosphate‑<i>N</i>‑oxide with Lysine 5,6-Aminomutase: Enzyme Flexibility toward Cofactor Analog

Lysine 5,6-aminomutase (5,6-LAM) is a 5′-deoxyadenosylcobalamin and pyridoxal-5′-phosphate (PLP) codependent radical enzyme that can accept at least three substrates, d-lysine, l-β-lysine and l-lysine. The reaction of 5,6-LAM is believed to follow an intramolecular radical rearrangement mechanism involving formation of a cyclic azacyclopropylcarbinyl radical intermediate (I^•). Similar I^•s are also proposed for other radical aminomutases, such as ornithine 4,5-aminomutase (4,5-OAM) and lysine 2,3-aminomutase (2,3-LAM). Nevertheless, experimental proof in support of the participation of I^• have been elusive. PLP is proposed to lower the energy of this elusive I^• by captodative stabilization and spin delocalization. In this work, we employ PLP-<i>N</i>-oxide (PLP-NO) to investigate the flexibility of 5,6-LAM toward cofactor analog and participation of I^• in the reaction mechanism. Our calculations show that substitution of PLP-NO for PLP stabilizes I^• by 35.2 kJ mol^–1 as a result of enhanced spin delocalization, which becomes the lowest energy state along the reaction sequence. Kinetic parameters and spectroscopic observations for PLP-NO similar to those of PLP demonstrate that PLP-NO mimics natural cofactor for 5,6-LAM. Interestingly, the flexibility of 5,6-LAM toward cofactor analog PLP-NO makes it an even more promising candidate for biocatalytic applications. Expectedly, the catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) is reduced by ∼3 times with PLP-NO as a cofactor. Various factors, including higher stabilization of proposed corresponding I^• for PLP-NO than that of PLP, could lead to the decrease in activity