Tadahiko Kajiwara

Biosynthetic pathway for C6-aldehydes formation from linolenic acid in green leaves

Abstract ‘Green odour’ of green leaves consists of six volatile compounds of C6-aldehydes and C6-alcohols including leaf aldehyde, (2E)-hexenal, and leaf alcohol, (3Z)-hexenol. These volatile compounds are biosynthesized from linolenic and linoleic acids via their respective hydroperoxides in green leaves. Four enzymes catalyse this biosynthetic pathway. Among these, especially interesting enzymes are lipoxygenase and hydroperoxide lyase. The enzyme system is found in green leaves bound to the lamella membrane of chloroplasts. The lipoxygenase stereoselectively adds oxygen to unsaturated fatty acids having a (1Z, 4Z)-pentadiene moiety, such as linolenic and linoleic acids, to produce 13-(S)-hydroperoxides. The hydroperoxide lyase cleaves the bond between C-12 and C-13 of these hydroperoxides into C6-aldehydes. With regard to environmental effects on these enzymes, the activities change seasonally and appear to be closely related to temperature and to solar radiation received.

Bell pepper fruit fatty acid hydroperoxide lyase is a cytochrome P450 (CYP74B)

Fatty acid hydroperoxide lyases cleave a C−C bond adjacent to a hydroperoxide group in lipoxygenase derived lipid hydroperoxides to form short‐chain aldehydes and oxo‐acids. Previously, we showed that fatty acid hydroperoxide lyase from bell pepper fruits is a heme protein whose spectrophotometric properties greatly resemble a cytochrome P450. In order to ascertain the relationship of it to the P450 gene family, we have cloned cDNA encoding fatty acid hydroperoxide lyase from immature bell pepper fruits. The cDNA encodes 480 amino acids, and shares homology with P450s mostly at the C terminus. The heme binding cysteine is recognizable at position 441. The most closely related P450 is allene oxide synthase (CYP74A), with which it has 40% identity. It qualifies the lyase as a member of a new P450 subfamily, CYP74B. From this finding, the enzyme is thought to be a novel member of P450 specialized for the metabolism of lipid peroxides.

Fatty acid 9- and 13-hydroperoxide lyases from cucumber1

Fatty acid hydroperoxide lyase (HPL) is a novel P‐450 enzyme that cleaves fatty acid hydroperoxides to form short‐chain aldehydes and oxo‐acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction‐based cloning strategy, we isolated two HPL‐related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13‐hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9‐ and 13‐hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS.

Fatty acid hydroperoxide cleaving enzyme, hydroperoxide lyase, from tea leaves

Abstract Hydroperoxide cleaving enzyme, hydroperoxide lyase (HPO lyase), was firstly purified to homogeneity from membranous fraction of tea leaves. The enzyme was separated with hydroxyl apatite gel chromatography into two fractions, HPO lyase I and II having Mr of 55 000 and 53 000, respectively. Almost the same pH-activity profiles were obtained with these two forms of HPO lyases. Antioxidants which are known as potent inhibitors for lipoxygenase such as nor-dihydroguaiaretic acid and butylated hydroxyanisole inhibited the activity of HPO lyase I. Activity for 13-hydroperoxide of α-linolenic acid was ca 10 times higher than that for 13-hydroperoxide of linoleic acid but the positional isomer, 13-hydroperoxide of γ-linolenic acid was a relatively poor substrate.

A lipid-hydrolysing activity involved in hexenal formation.

Short-chain aldehydes such as (3Z)-hexenal and n-hexanal are formed from lipids through sequential actions of lipid-hydrolysing, lipoxygenase and fatty acid hydroperoxide lyase activities. The aldehydes are formed upon wounding of plant tissues, and are reported to have bactericidal and fungicidal activities. Furthermore, it has been reported that the aldehydes can induce expression of a subset of genes involved in disease resistance and that they are involved in a defence response against insect herbivores. Although several genes encoding lipoxygenases and the lyases have been isolated, and characterized to some extent, only little is known about the enzyme accountable for the lipid-hydrolysing step. In this study, we tried to characterize the lipid-hydrolysing activity involved in the short-chain aldehyde formation in Arabidopsis. When Arabidopsis leaves were homogenized, (3Z)-hexenal was formed rapidly within a few minutes. During this time period, the amount of alpha-linolenic acid and C(16:3) rapidly decreased. Such a rapid increase of the aldehyde was repressed almost completely when the leaves were homogenized under a nitrogen stream, and instead free trienoic acids accumulated. A lipase inhibitor, quinacrine, successfully repressed the hydrolysis. It was revealed that trienoic acids in monogalactosyldiacylglycerol were predominantly hydrolysed during the formation of short-chain aldehydes. Collectively, it is suggested that the lipolytic enzyme involved in the short-chain aldehyde formation is a galactolipid-specific lipase.

Biosynthetic pathway of cucumber alcohol: Trans-2,cis-6-nonadienol via cis-3,cis-6-nonadienal

Abstract cis -3, cis -6-Nonadienal and cis -3-nonenal in Cucumis sativus were identified by comparison with synthetic specimens. The identification of these compounds, combined with biochemical evidence, suggests that cucumber alcohol and trans -2-nonenol are biosynthesized via cis -3-unsaturated aldehydes from linolenic and linoleic acid, respectively.

Distribution of lipoxygenase and hydroperoxide lyase in the leaves of various plant species

Abstract The enzyme activity responsible for volatile C 6 -aldehyde formation was accompanied by lipoxygenase and hydroperoxide lyase in the green leaves of 28 plant species tested, but the level of each enzymes activity varied. Lipoxygenase activity rather than hydroperoxide lyase activity appears to affect the overall C 6 -aldehyde formation. There was a positive correlation ( r = 0.712) between hydroperoxide lyase activity and the chlorophyll content of the green leaves; no correlation was found between lipoxygenase activity and chlorophyll content.

Biosynthesis of trans-2-hexenal in chloroplasts from Thea sinensis

Abstract The biosynthetic pathway of trans-2-hexenal, leaf aldehyde, in isolated chloroplasts of Thea sinensis leaves. was examined using a tracer experiment. A high and specific incorporation of radioactivity into cis-3-hexenal and trans-2-hexenal, was observed when linolenic acid-[U-14C] was incubated with the isolated chloroplasts. Thus, trans-2-hexenal was biosynthesized via cis-3-hexenal from linolenic acid in the chloroplasts.

Fatty acid hydroperoxide lyase in tomato fruits: cloning and properties of a recombinant enzyme expressed in Escherichia coli.

Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a λmax at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.

Distribution of an enzyme system producing cis-3-hexenal and n-hexanal from linolenic and linoleic acids in some plants

Abstract The activity of the enzyme system (E 2 -I) producing C 6 -aldehydes from C 18 -unsaturated fatty acids was investigated using about 40 plants. Green leaves of dicotyledonous plants belonging to the Sphenopsida, Pteropsida Theaceae and Leguminosae showed a high enzyme (E 2 -I) activity but edible leafy vegetables and fuits and monocotyledonous plants showed a low activity. Seasonal changes in the enzyme (E 2 -I) activities were observed. The concentrations of cis -3-hexenol (leaf alcohol) and trans -2-hexenal (leaf aldehyde) and the enzyme (E 2 -I) activities showed a correlation; high concentrations were observed in the summer but they were low in the winter.