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5-Lipoxygenase is the key enzyme in the formation of leukotrienes, which are potent lipid mediators of asthma pathophysiology. This enzyme translocates to the nuclear envelope in a calcium-dependent manner for leukotriene biosynthesis. Eight green fluorescent protein (GFP)-lipoxygenase constructs, representing the major human and mouse enzymes within this family, were constructed and their cDNAs transfected into human embryonic kidney 293 cells. Of these eight lipoxygenases, only the 5-lipoxygenase was clearly nuclear localized and translocated to the nuclear envelope upon stimulation with the calcium ionophore A23187. The N-terminal “β -barrel” domain of 5-lipoxygenase, but not the catalytic domain, was necessary and sufficient for nuclear envelope translocation. The GFP-N-terminal 5-lipoxygenase domain translocated faster than GFP-5-lipoxygenase. β-Barrel/catalytic domain chimeras with 12- and 15-lipoxygenase indicated that only the N-terminal domain of 5-lipoxygenase could carry out this translocation function. Mutations of iron atom binding ligands (His550 or deletion of C-terminal isoleucine) that disrupt nuclear localization do not alter translocation capacity indicating distinct determinants of nuclear localization and translocation. Moreover, data show that GFP-5-lipoxygenase β-barrel containing constructs can translocate to the nuclear membrane whether cytoplasmic or nuclear localized. Thus, the predicted β-barrel domain of 5-lipoxygenase may function like the C2 domain within protein kinase C and cytosolic phospholipase A2with unique determinants that direct its localization to the nuclear envelope.

Mutant and chimeric lipoxygenases were expressed in human embryonal kidney 293 cells to assess the importance of amino acids and domains for catalytic activity and positional specificity of molecular oxygen insertion. Histidines 360, 365, and 540, when changed to glutamine residues, completely abolished human platelet 12‐lipoxygenase activity. Altered histidines at positions 355, 383, and 392 retained enzymatic activity. The former three histidines could possibly serve as ligands for the catalytically essential non‐heme iron atom. Amino acids adjacent (residues 398‐417) to the five centrally located histidines conserved among all plant and animal lipoxygenases controlled to a limited extent the positional specificity of oxygenation of 12‐lipoxygenase. Variant A417I and the triple variant K416Q/A417I/V418M, designed to introduce 15‐lipoxygenase substitutions, transformed the platelet 12‐lipoxygenase that synthesizes exclusively 12‐hydro(pero)xy‐eicosatetraenoic acid (12‐H(P)ETE) to an enzyme capable of 10‐20% 15‐lipoxygenation. When all amino acids between positions 398‐429 of 12‐lipoxygenase had the corresponding 15‐lipoxygenase sequence, the enzyme made 66% 15‐lipoxygenase products. The latter enzyme had markedly reduced enzyme activity, though, indicating an apparent shift in the optimal alignment of substrate at the active site for hydrogen atom abstraction. The platelet enzyme could not be altered to form 5‐lipoxygenase products by similar manipulations of sequence within this region. Chimeric enzymes consisting of an NH2‐terminal segment from one lipoxygenase and the COOH terminus from another lipoxygenase or large substitutions resulted in nonfunctional enzymes. NH2‐terminal extensions, but not short deletions, could be tolerated functionally. These studies provide some new insights into lipoxygenase structure‐function in the absence of an unresolved three‐dimensional structure.—Chen, X.‐S., Funk, C. D. Structure‐function properties of human platelet 12‐lipoxygenase: chimeric enzyme and in vitro mutagenesis studies. FASEB J. 7:694‐701; 1993.

5-Lipoxygenase catalyzes the first two steps in the biosynthesis of leukotrienes, potent extracellular mediators of inflammation and allergic disorders. The unanticipated observation of 5-lipoxygenase in the nucleus of some cell types including bone marrow-derived mast cells (Chen, X. S., Naumann, T. A., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1995) J. Biol. Chem.270, 17993–17999) has raised speculation about intranuclear actions of leukotrienes or the enzyme itself. To explore the entry of 5-lipoxygenase into the nucleus we have transfected various cell types with expression vectors encoding native 5-lipoxygenase and green fluorescent protein/5-lipoxygenase (GFP-5LO) fusion proteins. 5-Lipoxygenase and green fluorescent protein/5-lipoxygenase co-localized with the nuclear DNA stain Hoechst 33258 in each cell type. The three main basic regions of 5-lipoxygenase were incapable of acting as “classical” nuclear localization signal sequences. Mutations that abolished enzyme activity/non-heme iron resulted in proteins that would no longer enter the nucleus. An NH2-terminal 5-lipoxygenase fragment of 80 residues was sufficient for directing nuclear localization of green fluorescent protein but not cytosolic pyruvate kinase. The combined data suggest that 5-lipoxygenase enters the nucleus not by a classical nuclear localization signal but by a non-conventional signal located in the predicted β-barrel domain that may be masked by structural alterations.