Helical remodeling augments 5-lipoxygenase activity

Abstract

The synthesis of pro-inflammatory 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-2 (H2) plugged the substrate access portal. Here, we present a new conformation of 5-LOX where H2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. 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 H2 designed to unlock the closed conformation and elongate H2 resulted in improved kinetic parameters, altered limited-proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active 5-LOX enzyme to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated H2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted, open conformation.