Reaction of Pyridoxal-5′-phosphate-N-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-beta-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-center dot). Similar I(center dot)s are also proposed for other radical aminomutases, such as ornithine 4,5-aminomutase (4,5-0AM) and lysine 2,3-aminomutase (2,3-LAM). Nevertheless, experimental proof in support of the participation of I-center dot have been elusive. PLP is proposed to lower the energy of this elusive I-center dot by captodative stabilization and spin delocalization. In this work, we employ PLP-N-oxide (PLP-NO) to investigate the flexibility of 5,6-LAM toward cofactor analog and participation of I-center dot in the reaction mechanism. Our calculations show that substitution of PLP-NO for PLP stabilizes I-center dot 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 (k(cat)/K-m) is reduced by similar to 3 times with PLP-NO as a cofactor. Various factors, including higher stabilization of proposed corresponding I-center dot for PLP-NO than that of PLP, could lead to the decrease in activity.