Purification and characterization of recombinant human neutrophil leukotriene B₄ ω-hydroxylase (cytochrome P450 4F3)

Recombinant human neutrophil leukotriene B₄ (LTB₄) ω-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14.8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the ω-hydroxylation of LTB₄ with a K(m) of 0.64 μM and V(max) of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH- P450 reductase and cytochrome b₅. Furthermore, various eicosanoids such as 20-hydroxy-LTB₄, 6-trans-LTB₄, lipoxin A₄, lipoxin B₄, 5-HETE and 12- HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently ω- hydroxylated, although their K(m) values were much higher than that of LTB₄. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A₁, and prostaglandin E₁, all of which are excellent substrates for the CYP4A fatty acid ω-hydroxylases. This is the first time human neutrophil LTB₄ ω-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity.