Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction

Abstract

The oxygenation reactions of polyunsaturated fatty acids include several examples in which different enzymes collaborate in synthesis of the end product. The cyclooxygenases are paired with cytochrome P450s in the transformation of arachidonic acid to prostaglandin (PG) endoperoxides and on to thromboxane A 2 and prostacyclin ( 1, 2 ). A P450 and cyclooxygenase participate in the opposite order in the metabolism of P450-derived 8,9-epoxyeicosatrienoic acid (8,9-EET) by cyclooxygenase, giving epoxy-hydroxy products ( 3 ). The plant pathway of jasmonic acid synthesis involves initial oxygenation by a 13 S -lipoxygenase (LOX) followed by transformation to an allene oxide by the P450, CYP74 ( 2, 4 ). Other examples include the cooperation of LOX and peroxygenase enzymes in the formation of epoxy and hydroxy fatty acids ( 5-7 ). The present work describes a new type of interaction, not so far described in nature, yet with the potential to produce fatty acid hydroxy-endoperoxides. Further transformations could readily form triols or other derivatives.The impetus for the present work arose from further studies of fatty acid epoxidation by catalase-related enzymes primed with an oxygen donor, analogous to an earlier report of arachidonate and 8-HETE epoxidation by catalase-related allene oxide synthase ( 8 ). One example involved enzymatic epoxidation of 13 S -hydroxy-␣ -linolenic acid on the 15,16-double bond. To assign the confi guration of the new 15,16-epoxide group, we proposed a scheme using soybean LOX to introduce a 13 S -hydroperoxide into Abbreviations: AD1/AD2, 15,16-epoxy-linolenate enantiomers eluted from Chiralpak AD chiral column; EET, epoxyeicosatrienoic acid; HAc, glacial acetic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; mCPBA, meta-chloroperoxybenzoic acid; P450, cytochrome P450; PG, prostaglandin; RP-HPLC, reversed phase HPLC; S N i, intramolecular nucleophilic substitution; SP-HPLC, straight phase HPLC; TPP, triphenylphosphine.

Oxidation of C18 Hydroxy‐Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase‐Related Hemoproteins Activated with Iodosylbenzene

“…(Note that the early-eluting peaks in Fig. 3 are irrelevant to this analysis: they are fatty acid endoperoxides resulting from the further transformation of 13 S -hydroperoxy-15,16-epoxides as characterized previously [21]). The results in Fig.…”

“…In addition to hydro(pero)xy-epoxy α-linolenic acid derivatives, monocyclic endoperoxide side-products formed and are reported in detail by Teder et al [21]. Enzymatically derived 15 S ,16 R - and 15 R ,16 S -epoxy-13 S -hydroxy derivatives were co-analyzed with the product of Fg-cat from 13 S -HOTE-ω3/PhIO on SP-HPLC using a Beckman 5-µm silica column (25 × 0.46 cm) and a solvent of hexane/isopropanol (100:5, v/v) with a flow rate at 1 mL/min (Results).…”

“…15 S ,16 R - and 15 R ,16 S -epoxyoctadeca-9 Z ,12 Z -dienoic acids were individually incubated with soybean 13 S -lipoxygenase in the presence of reducing agent SnCl 2 as described previously [21]. In addition to hydro(pero)xy-epoxy α-linolenic acid derivatives, monocyclic endoperoxide side-products formed and are reported in detail by Teder et al [21].…”

“…The products were analyzed as methyl esters and chromatographed on a Beckman silica column (0.46 × 25 cm) using a hexane/isopropanol (100:5; v/v) at a flow rate of 1 mL/min with the UV detection at 235 nm. Early-eluting products on chromatograms B and C are endoperoxide derivatives characterized by Teder et al [21]. …”

Synthesis of cyclooxygenase metabolites of 8,9-epoxyeicosatrienoic acid (EET): 11- and 15-hydroxy 8,9-EETs

“…10 Besides, EETs has been shown to be metabolized by CYP450, COX, LOX and other enzymes, producing secondary metabolites that have less well understood biological activity. For example, Teder et al 11 recently showed that 14,15-EET is a substrate for human 15-LOX-1 and murine 12-LOX, giving a mixture of isomers containing a hydroperoxy group at positions 5, 8 and 12. However, these metabolites have not been found in vivo yet and their biological significance remains unknown.…”