1 research outputs found
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<sup>•</sup>). Similar I<sup>•</sup>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<sup>•</sup> have
been elusive. PLP is proposed to lower the energy of this elusive
I<sup>•</sup> 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<sup>•</sup> in the reaction mechanism. Our calculations
show that substitution of PLP-NO for PLP stabilizes I<sup>•</sup> by 35.2 kJ mol<sup>–1</sup> 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><sub>cat</sub>/<i>K</i><sub>m</sub>) is reduced by ∼3
times with PLP-NO as a cofactor. Various factors, including higher
stabilization of proposed corresponding I<sup>•</sup> for PLP-NO
than that of PLP, could lead to the decrease in activity