Characterization of a C-5,13-Cleaving Enzyme of 13(S)-Hydroperoxide of Linolenic Acid by Soybean Seed

Abstract

~An activity was found in mature soybean seeds (Glycine max 1. cv Century) that cleaved 13(S)-hydroperoxy-9(2),11 (E),15(Z)-octadecatrienoic acid (1 3s-HPOT) into 13-0~0-9(Z),11(E)-tridecadienoic acid and two isomeric pentenols, Z(Z)-penten-l-ol and 1 -penten-3-ol. lsomeric pentene dimers were also produced and were presumably derived from the combination of two pentene radicals. 13(S)-Hydroperoxy-9(Z),ll(E)-octadecadienoic acid (1 3s-HPOD) was, by contrast, a poor substrate. Activity with 13s-HPOT increased 24-fold under anaerobic conditions reminiscent of a similar anaerobic promoted reaction of 13s-HPOD catalyzed by lipoxygenase (LOX) in the presence of linoleic acid. However, prior to ion-exchange chromatography, cleavage activity did not require linoleic acid. After separation by gel filtration followed by ionexchange chromatography, cleavage activity was lost but reappeared in the presence of either linoleic acid or dithiothreitol. Under these conditions cleavage activity was coincident with the activity of types 1 and 2 LOX. LOX inhibitors suppressed the cleavage reaction in a manner similar to inhibition of LOX activity. Heat-generated alkoxyl radicals derived from either 13s-HPOT or 13s-HPOD afforded similar products and yields of 13-0x0-9(Z),11 (.€)-tridecadienoic acid compared t o the enzymic reaction.The product 1-penten-3-01 may be the precursor of the "raw-bean" volatile ethylvinylketone.A hydroperoxide lyase, which cleaves between the hydroperoxide-bearing carbon and a vicinal methylene, was first described in mushroom (Wurzenberger and Grosch, 1984), and subsequently a similar enzyme was found in algae and one species of grass (see review by Gardner, 1991). This type of enzyme cleaves 13s-HPOT into 13-OTA and either pentene or pentenol, giving products similar to those from homolytic cleavage of 13s-HPOT. However, LOX, the enzyme catalyzing the oxidation of polyunsaturated fatty acids to hydroperoxides, can catalyze such a homolytic chain cleavage under anaerobic conditions (Garssen et al., 1971). Earlier, one of us reported the formation of 13-OTA from 13s-HPOT by a soybean (Gycine max L.) preparation and postulated that an anaerobic reaction of LOX was responsible . After submission of this paper, Kondo et al. (1995) "homolytic hydroperoxide lyase" in soybeans that catalyzes formation of 13-OTA and 2-penten-1-01 from 13S-HPOT, but these workers did not attempt to characterize the enzyme(s) involved. They found that 13-OTA possessed antifungal activity, possibly explaining a physiological function for the cleavage reaction.In this communication we characterize an activity in soybeans that cleaves 13s-HPOT into 13-OTA and two isomeric pentenols in a much higher yield than cleavage of 13s-HPOD. Although the reaction did not appear to require a LOX substrate (polyunsaturated fatty acid), it was stimulated by anaerobic conditions. After enzyme purification by ion-exchange chromatography, activity was lost, but it could be restimulated by either linoleic acid or DTT. Enzyme characterization of purif...

A Multifunctional Lipoxygenase with Fatty Acid Hydroperoxide Cleaving Activity from the Moss Physcomitrella patens

“…The present study is to our knowledge the first to describe a LOX/lyase/hydroperoxidase activity within a single cloned enzyme. Our results support the findings obtained with crude preparations or partially purified LOX that these enzymes have a multiple potential to transform hydroperoxide fatty acids (8,28,29). The PpLOX has a relaxed substrate tolerance and exhibits a remarkable diverse product spectrum.…”

“…Few LOX with lyase activity have been described that release ␣,␤,␥,␦-unsaturated aldehydes and a second alcohol fragment from fatty acids like it was observed with the PpLOX1 (28,29). Glasgow et al (8) investigated a preparation of porcine leukocytes that converts arachidonic acid to a mixture of products derived via 5-, 12-and 15-LOX reactions and also identified 12-ODTE as a proposed cleavage product of 12-HPETE.…”

Changes in Volatile Profiles and Activity of Hydroperoxide Lyase and Alcohol Dehydrogenase During the Development of Cabernet Sauvignon Grapes (Vitis vinifera L.)

“…Generally speaking, the content of C5 maintained a low level in all three tissues, which may be a result of the weak activity of 13-HPL. C5 volatiles were synthesised by the homolytic mechanism of 13-HPL (Grechkin, 1998;Salch et al, 1995. ), while C5 was more abundant in the leaves than in the grape skins and flowers.…”

Fatty acid hydroperoxides biotransformation by potato tuber cell-free extracts

“…Or, as suggested by Salch et al 1995, a secondary activity of Lox could explain the homolytic cleavage of HPO. This assumption was strengthened by the study of Nunez et al (2000) on the microalgae species Chlorella.…”

“…In starfish oocytes, the homolytic cleavage of arachidonic acid hydroperoxide gives a C-7 hydroxyacid and C-13 aldehydes (Brash et al 1991). Here are only a few reports of the occurrence of such enzymatic activities in higher plants, in olive oil (Angerosa et al 1998) and in soybean (Kondo et al 1995, Salch et al 1995, but the enzyme responsible for their synthesis is still unknown. Nevertheless, as reported by Table 3.…”