Elucidation of allosteric behavior and enzyme-effector complexes of human 15-lipoxygenase-2 through hydrogen-detuerium exchange mass spectrometry

Abstract

The enzymatic activity of human 15-lipoxygenase-2 (15-LOX-2) has been implicated in human inflammatory diseases such as atherosclerotic cardiovascular disease and plays an important role in maintaining homeostasis. 15-LOX-2 is one of six human lipoxygenase enzyme that catalyzes the (per)oxidation of fatty acids to produce both pro- and anti-inflammatory cellular signaling molecules.1 Despite its importance, current anti-inflammatories available on the market do not target lipoxygenase derived inflammation. There are three types of regulation of 15-LOX-2 activity: small allosteric regulators, selective inhibitors, and protein-protein/protein-membrane interactions. The first of these types of regulation is the focus of this study. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) allows for structural analysis of the complex formed with small allosteric regulators and selective inhibitors of 15-LOX-2. Using HDX-MS, we have shown excellent coverage of the primary sequence of 15-LOX-2 with dynamic properties comparable to previous reports. Further, in the presence of oleyl sulfate, a small molecule effector, regions of the protein were resolved to show alterations in protein flexibility. This data provides a basis for 15-LOX-2 regulation.