The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A₄ epoxide which is converted to a chemotactic leukotriene B₄ (LTB₄) by leukotriene A₄ hydrolase (LTA₄H). In addition, LTA₄H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA₄H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA₄H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA₄H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB₄ and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.

Understanding enzyme function requires characterizing not only static structure but also dynamics and ligand interactions. NMR spectroscopy provides this insight at atomic resolution, yet for large proteins the difficulty of resonance assignment has largely confined such studies to systems below ∼ 50 kDa, or to observing only methyl groups. Here we present an integrated magic-angle spinning (MAS) and solution NMR study of the 134 kDa tetrameric malate dehydrogenase from Ignicoccus islandicus ( Ii MDH), an enzyme of particular interest as an evolutionary intermediate between allosteric lactate dehydrogenases and non-allosteric malate dehydrogenases. By combining high-dimensional (up to 4D) MAS NMR experiments on sedimented protein with solution NMR, we achieved 92% backbone heavy-atom assignment and 91% assignment of all Ile-δ ₁ , Leu-δ ₁ /-δ ₂ , Val-γ ₁ /-γ ₂ , Met-ɛ and Thr-γ methyl groups. Building on these assignments, MAS NMR ¹⁵ N rotating-frame relaxation ( R _{1 p} ) measurements revealed pronounced microsecond-timescale backbone dynamics in functionally critical regions, including the catalytic loop and the mobile surface loop. Complementary methyl-axis order parameters from solution NMR identified additional flexible sites in the hydrophobic core. Chemical shift perturbation experiments upon addition of the substrate analogue oxamate, monitored via backbone ¹ H- ¹⁵ N TROSY, revealed both active-site contacts and rearrangements of helices a2F and a3G, regions implicated in allosteric signal transmission. The integrated approach demonstrated here exploits the distinct strengths of MAS and solution NMR, and provides a comprehensive view of structure, dynamics, and substrate interactions in a large oligomeric enzyme that would not be accessible by either technique alone.