Lipoxygenases (LOXs) are a family of non-heme, metal containing enzymes that catalyze the oxidation of poly unsaturated fatty acids. The resulting hydroperoxide products are key signaling molecules that are involved in seed germination, development and pathogen defense in plants and in both normal homeostasis and pro-inflammatory pathways in human LOXs. Previous kinetic studies for model plant (soybean lipoxygenase, SLO) and animal (human epithelial 15-LOX-2) 15-LOX isozymes have implicated substrate selectivity regulation by fatty acid effectors. The location of effector binding and the mechanism by which fatty acid effectors allosterically modulate substrate binding and selectivity at the active site is not fully resolved. We present results from a combination of biophysical tools, including dynamic light scattering, differential scanning calorimetry, and isothermal titration calorimetry to demonstrate effector binding and show that fatty acid allostery in the monomeric 15-LOXs is not controlled through oligomerization or large-scale conformational changes. Further, temperature-dependent hydrogen-deuterium exchange mass spectrometry (TD-HDXMS) reveals comparable allosteric networks in SLO and 15-LOX-2 that show altered flexibility of helix α2, which acts as a gate to the substrate entrance. Taken together, these results provide evidence to support the allosteric regulation of these enzymes is controlled through the dynamics of helix α2. Kinetic analysis of site-directed LOX variants to target residues validate the <i>in silico</i> docking model of the fatty acid allosteric sites in these model plant and animal 15-LOXs.

Dynamic allostery networks in plant and animal 15-lipoxygenases

ACS Spring 2022

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