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 (KKK653-655) 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 KKK653-655 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.