Leukotriene A synthase activity of purified mouse skill arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfe ction of its cDNA in pEF-BOS carrying an elongation factor-1 alpha promoter . When crude extract of the transfected COS-7 cells was incubated with arac hidonic acid, 8-hydroxy-5,9,11,14-eicosatetraenoic acid was produced as ass essed by reverse- and straight-phase high performance liquid chromatographi es. The recombinant enzyme also reacted on a-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaeno ic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction r ates of arachidonic acid, respectively. In contrast, linoleic acid was a po or substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also exp ressed in an Escherichia coli system using pQE-32 carrying six histidine re sidues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 mu mol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymat ic properties of the purified 8-lipoxygenase were essentially the same as t hose of the enzyme expressed in COS-7 cells. When the purified X-lipoxygena se was incubated with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, two ep imers of 6-trans-leukotriene B-4, degradation products of unstable leukotri ene A(4), were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A(4) from 5-hydropero xy fatty acid. Reaction rate of the leukotriene A synthase was approximatel y 7% of arachidonate 8-lipoxygenation. In contrast to the linear time cours e of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation. (C) 19 99 Elsevier Science B.V. All rights reserved.