Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential

Abstract

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2’s reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals

“…Measurements of kinetic characteristics of AA-PE oxidation vs free AA oxidation by 15LO1 and 15LO2 in the presence and absence of human PEBP1 for both enzymes, showed that the action of PEBP1 was unidirectional although the effects were more robust for 15LO2 than 15LO1 (Table S1). Vmax, Km, Kcat and Kcat/Km for free AA as a substrate in the absence of PEBP1 were in reasonable agreement with the previously published data for 15LO2 (Green et al, 2016). When PEBP1 was added to 15LO2, we found no significant changes in Vmax, a 1.7-fold increase in Km and a slight decrease of Kcat/Km for free AA.…”

Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

“…These values indicate that DHA has the fastest rate of substrate capture at low substrate concentrations, kcat/KM, but 7S-HDHA has the fastest rate of product release at high substrate concentrations, kcat (49). For comparison, the kinetic parameters for h15-LOX-2 and AA are 0.96 sec -1 for kcat and 0.19 sec -1 µM -1 for kcat/KM, similar to published values (15,43), indicating that at low substrate concentration, 7S-HDHA is a comparable substrate to that of AA, but DHA is preferred. These data support Pathway 5 (Scheme 1) as the most likely in vitro biosynthetic route for RvD5 production.…”

“…Previously, it was shown that h15-LOX-2 oxygenated 5S-HpETE, but not 12S-HpETE or 15S-HpETE (15). Together with the current work, this suggests that the h15-LOX-2 active site tolerates substrates which are oxygenated close to the carboxylate end, but not those substrates oxygenated closer to the methyl end.…”

“…In fact, 7S-HDHA and 7S-HpDHA are viable substrates of h15-LOX-2, with greater kcat values but lower kcat/KM values than DHA, making them less reactive substrates than DHA at low concentration with h15-LOX-2. Previous work comparing the reaction of AA and 5S-HpETE with h15-LOX-2 demonstrated a similar trend, with 5S-HpETE having a greater kcat but a smaller kcat/KM than AA (15). Considering that hydrogen atom abstraction is generally the first irreversible ratedetermining step for kcat and kcat/KM for fatty acid substrates, these results suggest that the rate of oxylipin binding is markedly slower relative to fatty acid binding for h15-LOX-2 (15).…”

“…They are named for their ability to stereo-specifically oxygenate at C15 of arachidonic acid (AA) (10). h15-LOX-1 converts AA into a mixture of approximately 90% 15(S)hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15S-HpETE) and 10% 12(S)-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HpETE), while h15-LOX-2 oxygenates substrates with strict regiospecificity (15), producing only 15-HpETE from AA. h15-LOX-1 is implicated in many diseases, such as inflammation, stroke, colorectal cancer, and atherosclerosis (11), while h15-LOX-2 has only recently been identified as playing a role in atherosclerosis and inflammation (4,16).…”