Elucidating the Open and Active Conformation of Human 5‐Lipoxygenase

Abstract

Human 5‐Lipoxygenase (5‐LOX) is an enzyme that responds to the call for inflammation by initiating the synthesis of leukotrienes involved in asthmatic and allergic reactions and acts to sustain inflammatory reactions. Acting at the nuclear membrane in leukocytes, 5‐LOX oxygenates its lipid substrate arachidonic acid to leukotriene A4. The structure of 5‐LOX was solved in a closed conformation with helix‐α2 shortened and two large hydrophobic residues from the helix corking the active site. Most other animal lipoxygenase structures were solved with an elongated helix‐α2 allowing for an open conformation of the active site. Our goal is to study the molecular determinants important for the transition from a closed to open structure. We used site‐directed mutagenesis of helix‐α2 residues to favor either a more opened or closed conformation. We also used inhibitors to probe the conformational flexibility of 5‐LOX by testing susceptibility to cleavage by limited proteolysis and changes in melting temperature by differential scanning fluorimetry. So far, we have observed that by mutating key residues to encourage a closed conformation, 5‐LOX was less susceptible to proteolysis and melted at a higher temperature. We also showed that by mutating the same residues to encourage an open conformation, 5‐LOX was more susceptible to proteolysis. When bound to a competitive inhibitor nordihydroguaiaretic acid (NDGA), 5‐LOX melts at significantly lower temperatures and appears more susceptible to proteolytic cleavage. However, mutations that stabilize the closed conformation reduce these NDGA‐induced effects. By enhancing the presence of a closed or open conformation by mutagenesis, our results continue to hint at the structural significance of the elongated helix‐α2 in 5‐LOX dynamics. Determination of a structure of 5‐LOX in an open conformation will enable understanding of substrate acquisition and inhibitor binding. An open conformation of 5‐LOX could aid in structure‐based drug design and would foster the development of new therapies for inflammatory disorders.