Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli.
Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.
Qiao
N
N
Department of Pharmacology, Kanazawa University School of Medicine, Kanazawa 920-8640, Japan.
Takahashi
Y
Y
Takamatsu
H
H
Yoshimoto
T
T
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000096:biosynthesis / Q000235:genetics*; D016718:Arachidonic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015289:Leukotrienes / Q000378:metabolism; D051379:Mice; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity
D018076:DNA, Complementary; D015289:Leukotrienes; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; C054192:arachidonate 8-lipoxygenase