Mechanism-based Inhibition Reveals Transitions between Two Conformational States in the Action of Lysine 5,6-Aminomutase: A Combination of Electron Paramagnetic Resonance Spectroscopy, Electron Nuclear Double Resonance Spectroscopy, and Density Functional Theory Study

An “open”-state crystal structure of lysine 5,6-aminomutase suggests that transition to a hypothetical “closed”-state is required to bring the cofactors adenosylcobalamin (AdoCbl) and pyridoxal-5′-phosphate (PLP) and the substrate into proximity for the radical-mediated 1,2-amino group migration. This process is achieved by transaldimination of the PLP–Lys144β internal aldimine with the PLP–substrate external aldimine. A closed-state crystal structure is not available. UV–vis and electron paramagnetic resonance studies show that homologues of substrate d-lysine, 2,5-DAPn, 2,4-DAB, and 2,3-DAPr bind to PLP as an external aldimine and elicit the AdoCbl Co–C bond homolysis and the accumulations of cob­(II)­alamin and analogue-based radicals, demonstrating the existence of a closed state. ²H- and ³¹P-electron nuclear double resonance studies, supported by computations, show that the position for hydrogen atom abstraction from 2,5-DAPn and 2,4-DAB by the 5′-deoxyadenosyl radical occurs at the carbon adjacent to the imine, resulting in overstabilized radicals by spin delocalization through the imine into the pyridine ring of PLP. These radicals block the active site, inhibit the enzyme, and poise the enzyme into two distinct conformations: for even-numbered analogues, the cob­(II)­alamin remains proximal to and spin-coupled with the analogue-based radical in the closed state while odd-numbered analogues could trigger the transition to the open state of the enzyme. We provide here direct spectroscopic evidence that strongly support the existence of a closed state and its analogue-dependent transition to the open state, which is one step that was proposed to complete the catalytic turnover of the substrate lysine

Tyrosine fluorescence spectra for the determination of Cu²⁺ binding affinity.

<p>(A) Aβ1-16, (B)Aβ1-24, (C)Aβ1-29, (D)Aβ1-35, (E)Aβ1-40. The concentration of Aβ peptides was 10 µM. The solid lines represent the best fitting curve using the independent two-Cu mode, whereas dot lines show the fitting curve simulated using one-Cu mode as depicted in the section of material and methods.</p

The TEM images of Aβ fibril morphologies.

<p>Images A, C, E and G represent the fibril morphologies for Aβ1-40, Aβ1-35, Aβ1-29 and Aβ1-16 with Cu²⁺ stripped off by EDTA, respectively. Images B, D, F and H represent the morphologies for Aβ1-40, Aβ1-35, Aβ1-29 and Aβ1-16 in the presence of Cu²⁺, respectively.</p

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

The aggregation profiles.

<p>The aggregation profile determined by turbidity assay in the absence (A) and the presence (B) of Cu²⁺ for different Aβ peptides, (⋄) Aβ1-16, (▾) Aβ1-24, (▴) Aβ1-29, (○) Aβ1-35, and (▪) Aβ1-40.</p

The plot of secondary structure content vs. Cu²⁺ concentration.

<p>The plot for different Aβ peptides, (▪) Aβ1-16, (♦) Aβ1-24, (▴) Aβ1-29, (○) Aβ1-35, (▾) Aβ1-40 and (A) β–sheet percentage, (B) random coil percentage, and (C) α-helx. (D) The plot of β–sheet propensity vs. Aβ peptides.</p

Circular dichroism spectra of Aβ peptides.

<p>CD spectra for different Aβ peptides, (▿) Aβ1-16, (□) Aβ1-24, (•) Aβ1-29, (○) Aβ1-35, (▪) Aβ1-40, in the absence (A) and presence (B) of Cu²⁺. The concentration for both Aβ peptides and Cu²⁺ used in measurements was 30 µM. A normalized root mean square standard deviation (NRMSD) parameter was introduced to indicate for the quality between observed and calculated CD spectra.</p

The content of secondary structure for the different Aβ peptides in the presence or absence of Cu²⁺ as calculated from CD spectra.

<p>*NRMSD (normalized root mean square standard deviation)  =  [(θ_obs(λ)-θ_cal(λ))²/(θ_obs(λ))²]^1/2.</p

The estimated EPR parameters of Aβ/copper (II) complex for the different Aβ peptides.

<p>The estimated EPR parameters of Aβ/copper (II) complex for the different Aβ peptides.</p

The estimated copper (II) binding constant using one-Cu and dependent two-Cu models for the different Aβ peptides.

<p>The estimated copper (II) binding constant using one-Cu and dependent two-Cu models for the different Aβ peptides.</p