Helical remodeling augments 5-lipoxygenase activity in the synthesis of proinflammatory mediators

The synthesis of proinflammatory leukotrienes implicated in asthma, allergic rhinitis, and atherosclerosis is initiated by the enzyme 5-lipoxygenase (5-LOX). The crystal structure of human Stable-5-LOX revealed a conformation where the catalytic iron was inaccessible to bulk solvent as two aromatic residues on a conserved helix-alpha 2 (H alpha 2) plugged the substrate access portal. Whether 5-LOX can also adopt a more open conformation has not been resolved. Here, we present a new conformation of 5-LOX where H alpha 2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. Our observation of the sigmoidal kinetic behavior of 5-LOX, which is indicative of positive cooperativity, is consistent with a substrate-induced conformational change that shifts the ensemble of enzyme populations to favor the catalytically competent state. Strategic point mutations along H alpha 2 designed to unlock the closed conformation and elongate H alpha 2 resulted in improved kinetic parameters, altered limited proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active Stable-5-LOX to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated H alpha 2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted open conformation. Taken together, these data provide valuable insights into the synthesis of leukotrienes.

Automated summary

This study reveals two distinct conformations of 5-lipoxygenase (5-LOX) in its catalytic reaction through structural and dynamic experiments. By strategic mutations, researchers successfully unlocked a more open conformation and improved the enzyme's activity. These findings are of great importance for understanding the synthesis mechanism of leukotrienes.