Abstract
Epidermal lipoxygenase-3 (eLOX3) exhibits hydroperoxide isomerase activity implicated in epidermal barrier formation, but its potential dioxygenase activity has remained elusive. We identified herein a synthetic fatty acid, 9E,11Z,14Z-20:3ω6, that was oxygenated by eLOX3 specifically to the 9S-hydroperoxide. Reaction showed a pronounced lag phase, which suggested that eLOX3 is deficient in its activation step. Indeed, we found that high concentrations of hydroperoxide activator (e.g. 65 μM) overcame a prolonged lag phase (>1 h) and unveiled a dioxygenase activity with arachidonic acid; the main products were the 5-, 9-, and 7-hydroperoxyeicosatetraenoic acids (HPETEs). These were R/S mixtures (ranging from ∼50:50 to 73:27), and as the bis-allylic 7-HPETE can be formed only inside the enzyme active site, the results indicate there is oxygen availability along either face of the reacting fatty acid radical. That the active site oxygen supply is limited is implied from the need for continuous re-activation, as carbon radical leakage leaves the enzyme in the unactivated ferrous state. An Ala-to-Gly mutation, known to affect the positioning of O(2) in the active site of other lipoxygenase enzymes, led to more readily activated reaction and a significant increase in the 9R- over the 5-HPETE. Activation and cycling of the ferric enzyme are thus promoted using the 9E,11Z,14Z-20:3ω6 substrate, by continuous hydroperoxide activation, or by the Ala-to-Gly mutation. We suggest that eLOX3 represents one end of a spectrum among lipoxygenases where activation is inefficient, favoring hydroperoxide isomerase cycling, with the opposite end represented by readily activated enzymes in which dioxygenase activity is prominent.
Highlights
Epidermal lipoxygenase-32 is one of the two lipoxygenases (LOX) involved in skin barrier formation [1, 2]
Trapping of the Primary Products—To focus on the primary LOX reaction, we identified the following three key factors that almost eliminated secondary product formation: (i) incubation at 0 °C preferentially reduces hydroperoxide isomerase activity compared with dioxygenase activity (28 –31); (ii) an excess of 15S-hydroperoxyeicosatetraenoic acids (HPETEs) included in the incubation competes with the conjugated triene hydroperoxide products for the hydroperoxide isomerase activity, and the excess 15S-HPETE helps keep the enzyme in the ferric state; and (iii), addition of 4-hydroxy-TEMPO (1.5 mM), a water-soluble antioxidant, prevents nonenzymatic lipid peroxidation without causing significant enzyme inhibition [32]
The usual aspects suggest that the basic machinery for LOX catalysis is fully functional in Epidermal lipoxygenase-3 (eLOX3), whereas the unusual aspects of eLOX3 point to the possibility that eLOX3 has limited oxygen access in the active site
Summary
Epidermal lipoxygenase-3 (eLOX3) is one of the two lipoxygenases (LOX) involved in skin barrier formation [1, 2]. In addition to its involvement in skin barrier formation, eLOX3 attracts attention because of its unusual enzymology It is classified as a LOX based on its amino acid sequence (e.g. 58% identity to 12R-LOX) yet its name eLOX3 lacks the usual designation such as 5-, 12-, or 15-LOX. To enter the catalytic cycle the ferrous enzyme has to be oxidized to the ferric form first, usually by 1 eq of their own hydroperoxide product This process, commonly known as the activation step, accounts for the initial lag phase in the progress curve of LOX reactions [12, 13]. Unlike typical LOX, eLOX3 does not require iron-reducing agents for its hydroperoxide isomerase activity That this reaction is catalysis, rather than a single turnover, implies that the ferrous state of eLOX3 is largely preserved. Higher than usual concentrations of fatty acid hydroperoxide might be
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