Alexander N. Grechkin

The lipoxygenase pathway in garlic (Allium sativum L.) bulbs" detection of the novel divinyl ether oxylipins

Incubations of [1‐14C]linoleic acid or [1‐14C]‐(9Z.11E, 13S)‐13‐hydropero xy‐9,11‐octadecadienoic acid (13‐HPOD) with juice of garlic bulbs lead to the formation of one predominant labelled product, viz., the novel divinyl ethery (9Z,11E, 1′E)‐12‐(1′‐hexenyloxy)‐9,11‐odecadienoic acid (‘etheroleic acid’). With lesser efficiency [1‐14C]α‐linolenic acid or [1‐14C](9Z,11E,13S,15Z)‐13‐hydroperoxy‐9,11,15‐octadecatrienoic acid (13‐HPOT) are converted in this way into (9Z,11E,1′E,3′Z)‐12‐(1′,3′‐hexadienyloxy)‐9,11‐dodecadienoic acid (‘etherolenic acid’). Thus, garlic bulbs possess the activity of a new 13‐hydroperoxide‐specific divinyl ether synthase.

Tomato CYP74C3 is a Multifunctional Enzyme not only Synthesizing Allene Oxide but also Catalyzing its Hydrolysis and Cyclization

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)‐hydroperoxide with dilute suspensions of LeAOS3 (10–20 s, 0 °C) yield mostly the expected allene oxide (12Z)‐9,10‐epoxy‐10,12‐octadecadienoic acid (9,10‐EOD), which was detected as its methanol‐trapping product. In contrast, the relative yield of 9,10‐EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80‐fold larger amounts of LeAOS3, while α‐ketol and the cyclopentenone rac‐cis‐10‐oxo‐11‐phytoenoic acid (10‐oxo‐PEA) became the predominant products. Both the α‐ketol and 10‐oxo‐PEA were also produced when LeAOS3 was exposed to preformed 9,10‐EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)‐hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.

Hydroperoxides of α-ketols

Metabolism of [1-14C]linolenic acid, 13-hydroperoxy-8(Z),11(E),15(Z)-[1-14C]octadecatrienoic acid (13-HPOT) and 9-hydroperoxy-10(E),12(Z),15(Z)-[1-14C]octadecatrienoic acid (9-HPOT) was studied by enzyme preparations from flax, wheat and corn seeds, containing two enzymes of fatty acid metabolism, namely, lipoxygenase and hydroperoxide dehydrase. Along with the previously known products of the hydroperoxide dehydrase pathway, the radiolabel was incorporated into some more polar metabolites. These polar products 1 and 4, formed from 13-HPOT and 9-HPOT, respectively, were purified by reversed-phase and normal-phase HPLC, and investigated by ultraviolet spectroscopy, chemical-ionization and electron-impact mass spectrometry, and 1H-NMR. The data obtained suggest that metabolites 1 and 4 are 9-hydroperoxy-12-oxo-13-hydroxy-10(E),15(Z)-octadecadienoic acid and 9-hydroxy-10-oxo-13-hydroperoxy-11(E),15(Z)-octadecadienoic acid, respectively. 12-oxo-13-hydroxy-9(Z),15(Z)-[1-14C]octadecadienoic acid (12,13-alpha-ketol) and 9-hydroxy-10-oxo-12(Z),15(Z)-[1-14C]octadecadienoic acid (9,10-alpha-keto) are the direct precursors of metabolites 1 and 4, respectively. Metabolites 1 and 4 are formed from the corresponding HPOT precursors in two stages; (a) conversion of hydroperoxide into the alpha-ketol by hydroperoxide dehydrase and (b) the lipoxygenase oxidation of the alpha-ketol. Different lipoxygenases were found to oxidize alpha-ketols. Oxidation of the 3(Z)-buten-1-onyl moiety of alpha-ketols presents an unusual and previously unknown type of lipoxygenase reaction.

Formation of ketols from linolenic acid 13-hydroperoxide via allene oxide. Evidence for two distinct mechanisms of allene oxide hydrolysis

Incubations of [1-14C]13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) with hydroperoxide dehydrase preparations from flax seeds lead to the formation of a novel ketol 2 along with the previously known 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic (12,13-alpha-ketol) and 9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic (gamma-ketol) acids. Compound 2 was identified as 11-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid (11,12-alpha-ketol) in accordance with the data of ultraviolet, mass (chemical ionization and electron impact) and 1H-NMR spectra. During long-term (30 min) incubations the yields of gamma-ketol and 11,12-alpha-ketol increased markedly and the yield of 12,13-alpha-ketol decreased in response to the pH change from basic (pH 7.4) to acidic (pH 5.8) conditions. Short-term (15 s) incubations of 13-HPOT with hydroperoxide dehydrase, terminated by HCl fixation, led to the formation of gamma-ketol and ketol 2. A similar incubation, followed by NaOH fixation, afforded only 12,13-alpha-ketol. The trapping of allene oxide (a primary product of hydroperoxide dehydrase) with pure methanol gives only compound 4 (12,13-alpha-ketol methyl ether). Products 5 (gamma-ketol methyl ether) and 6 (11,12-alpha-ketol methyl ether) were formed along with 4 as a result of trapping with acidified methanol. The results obtained indicate that: (a) the formation of 12,13-alpha-ketol is base-dependent; (b) the formation of gamma-ketol and ketol 2 is acid-dependent. Two distinct mechanisms of allene oxide hydrolysis are proposed: (1) nucleophilic (SN2 or SN1, OH- is an attacking group) substitution, resulting in formation of 12,13-alpha-ketol; (2) electrophilic (SE-like) reaction initiated by protonation of oxirane, affording gamma-ketol and 11,12-alpha-ketol.

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

Abstract The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH4 reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by 1H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [14C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.

Cyclization of natural allene oxide fatty acids. The anchimeric assistance of β,γ-double bond beside the oxirane and the reaction mechanism

Abstract Formation of cyclopentenones was followed from linoleic, α-linolenic and γ-linolenic acid hydroperoxides (HPOD, HPOT(α) and HPOT(γ), respectively) via allene oxides in the presence of flax seed allene oxide synthase. Although 13-HPOT(α) and 9-HPOT(γ) were effective cyclopentenone precursors, 13-HPOD, 9-HPOD(γ) and 9-HPOT(α) were not. These results suggest that the presence of a double bond in β,γ-position toward the hydroperoxide function causes the strong effect of anchimeric assistance, increasing the cyclization rate by 2–3 orders of magnitude. The minor 15( E ) isomer was formed from 13-HPOT along with usual 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA). The remarkable (about 2-fold) suppression of 12-oxo-PDA formation was observed under acidic (pH 5.5) conditions in comparison to the alkaline (pH 7.8) ones. The mechanism of double bond-assisted allene oxide cyclization, comprising dipolar pericyclic ring closure in zwitterionic intermediate, is proposed.

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site‐directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild‐type LeAOS3. Thus, both sites 295 and 297 localized within the “I‐helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

The lipoxygenase pathway in tulip (Tulipa gesneriana): detection of the ketol route

The in vitro metabolism of [1-(14)C]linoleate, [1-(14)C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (alpha-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (gamma-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15, 16-dehydro alpha- and gamma-ketols were the main metabolites of [1-(14)C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave alpha-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, gamma-ketol reductase, which reduces gamma-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-gamma-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-(14)C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (10(5) g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species.

DIVINYL ETHER SYNTHASE FROM GARLIC (ALLIUM SATIVUM L.) BULBS : SUB-CELLULAR LOCALIZATION AND SUBSTRATE REGIO- AND STEREOSPECIFICITY

Sub‐cellular localization and some properties of 13‐hydroperoxide‐specific divinyl ether synthase from garlic bulbs were studied. Sub‐cellular fractions from garlic bulbs were incubated with [1−14C](9Z,11E,13S)‐13‐hydroperoxy‐9,11‐octa‐decadienoic acid (13‐HPOD). The predominant part of divinyl ether synthase activity from garlic bulbs was found in the microsomal fraction. The enzyme utilizes 13(S)‐HPOD as its preferential substrate. Other hydroperoxides, including 9(S)‐HPOD, gave much poorer yields of divinyl ethers. Unreacted hydroperoxide after incubation of 13(R,S)‐HPOD with enzyme was composed of up to 94% 13(R)‐HPOD. Thus, divinyl ether synthase possesses stereoselectivity, utilizing preferentially the (S)‐enantiomer.

Formation of cyclopentenones from all-(E) hydroperoxides of linoleic acid via allene oxides. New insight into the mechanism of cyclization

Conversions of (Z,E)‐ and (E,E)‐isomers of linoleic acid 13‐ and 9‐hydroperoxides with flax and maize allene oxide synthase were studied. All‐(E) but not (Z,E) hydroperoxides readily undergo cyclization via allene oxides into trans‐cyclopentenones. These results suggest that double bond geometry dramatically affects the formation of pericyclic pentadienyl cation intermediate and thus the capability of 18:2‐allene oxides to undergo electrocyclization into cyclopentenones.