A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein

Leukotrienes (LT) are lipid mediators of the inflammatory response that play key roles in diseases such as asthma and atherosclerosis. The precursor leukotriene A₄ (LTA₄) is synthesized from arachidonic acid (AA) by 5‑lipoxygenase (5-LOX), a membrane-associated enzyme, with the help of 5‑lipoxygenase-activating protein (FLAP), a nuclear transmembrane protein. In lipoxygenases the main chain carboxylate of the C-terminus is a ligand for the non-heme iron and thus part of the catalytic center. We investigated the role of a lysine-rich sequence (KKK^653-655) 20 amino acids upstream of the C-terminus unique to 5-LOX that might displace the main-chain carboxylate in the iron coordination sphere. A 5-LOX mutant in which KKK^653-655 is replaced by ENL was transfected into HEK293 cells in the absence and presence of FLAP. This mutant gave ~20-fold higher 5-LOX product levels in stimulated HEK cells relative to the wild-type 5-LOX. Co-expression of the enzymes with FLAP led to an equalization of 5-LOX products detected, with wild-type 5-LOX product levels increased and those from the mutant enzyme decreased. These data suggest that the KKK motif limits 5-LOX activity and that this attenuated activity must be compensated by the presence of FLAP as a partner protein for effective LT biosynthesis.