Hydroperoxide dehydrase

Incubation of hydroperoxide dehydrase from com Zea mays L.) was found to catalyse the conversion of 13(S)-HPOD into an unstable allene oxide derivative, 12,13(8)-epoxylinolenie acid [12,13(S)-EOD] [32]. 

In addition to the higher plants mentioned above, other organisms are also known to possess the jasmonic acid pathway. The green alga Chlorella pyrenoidosa was examined for its ability to metabolise 13(S)-HPOD. The study showed that the Chlorella extracts possessed hydroperoxide dehydrase and other enzymes of the jasmonic acid pathway. However, under normal laboratory conditions for culture growth, neither jasmonic acid nor metabolites of the jasmonic acid pathway were present in Chlorella [40]. 

Substrate Specificity. Hydroperoxide dehydrase from sunflower cotyledons was about four-fold more active wiA 13-hydroperoxylinoleic acid Aan 13-hydroperoxylinolenic acid. In contrast, leaf hydroperoxide lyase was more Aan 10-fold more active wiA 13-hydroperoxylinolenic acid Aan wiA 13-hydroperoxylinoleic acid. However, root hydroperoxide lyase showed no preference between Ae two substrates, again suggesting Aat roots have a different form of Ae enzyme Aan leaves. NeiAer 9-hydroperoxylinoleic nor 9-hydroperoxylinolenic acids were suitable substrates for any of Ae hydroperoxide metabolizing enzymes in sunflower. 

Molecular Weight Estimation. The molecular weights of Ae hyAoperoxide lyase enzymes from leaves and roots were different. Root lyase was 122,000 Da, whereas Ae leaf lyase was larger at 178,000 Da. Hydroperoxide dehydrase from sunflower cotyledons had a molecular weight of 196,000 Da. Molecular weight standards were Ayroglobulin (669,000), apoferritin... 

Figure Distribution of hydroperoxide dehydrase and hydroperoxide lyase among organs in sunflower seedlings.

Figure 2. Ion exchange separation of sunflower cotyledon hydroperoxide dehydrase (HD) and hydroperoxide lyase (HL) at various plant ages.

The most recent findings relating to lipoxygenase pathway in plants concern allene oxides, the primary products of enzymatic transformations of fatty acid hydroperoxides. Spontaneous hydrolysis of allene oxides leads to the formation of a- and y-ketols [1]. It has been also found that hydroperoxide dehydrase from flax seeds converts 8-hydroxy-155-hydroperoxy derivatives of arachidonic and eicosapentaenoic acids into a-ketols and prostaglandin A3 analogues [2]. These data allow us to hypothesize formation of ketols of 9-hydroxy derivative of a-linolenic acid. 

Figure 1. The reversed-phase radio-HPLC analysis of the products from 9-hydroxy-16-hydroperoxy-10( ),12(Z),14( )-[l- C]octadecatrienoic acid incubation with the hydroperoxide dehydrase prepeu ation from com seeds. Incubation was performed at pH 7.5 (A) or 6.3 (B). Peak 4, 9-hydroxy-16-hydroperoxy-10( ),12(Z),14( )-octadecatrienoic acid peaks 1,2,3, metabolites 1,2,3, respectively.

The amount of this metabolite, retention time 31 min (compound 3, fig.lA) accounted for approximately 40% of total recovered radioactivity at pH 7.5. When incubation medium pH was 6.3 the amount of compound 2, retention time 27 min. (fig. IB) was the same as the yield of the main product (about 10%). The change of direction of the substrate metabolism corresponds to different formation of a- and y-ketols under weak acidic and neutral pH of incubation medium. The similar stimulation of y-ketol formation under weak acidic pH values have been observed in the studying of metabolism of a-linolenic acid hydroperoxides by hydroperoxide dehydrases from different plant species [3]. The UV spectrum of metabolite 2 exhibits a typical oxoene chromophore, at 227 nm. The data obtained allow us to suggest that compound 2 is y-ketol of 9-hydroxy derivative, 9,12-dihydroxy-15-oxo-10( ),13( )-octadecadienoic acid. 

The obtained results allow us to conclude that com seed hydroperoxide dehydrase catalyzes dehydration of hydroperoxide groups at C-16 position with formation of a- and y-ketols of 9-hydroxylinolenate. We hypothesize the formation of cyclopentenone 9-hydroxy-ll-[2-ethyl-3-oxo-cyclopent-4-enyl]-10( )-undecenoic acid. 

Hamberg M. Mechanism of com hydroperoxide dehydrase detection of 12,13(5)-oxido-9(Z),11-octadecadienoic acid. Biochim Biophys Acta 1987 920 76-84. 

In barley grains, lipoxygenase has been well characterized and two isoenzymes have been purified [1-3] but the characterization of hydroperoxide-metabolizing enzymes has received less attention. Hydroperoxide-consuming enzymes can be divided into two types of enzymes. Hydroperoxide lyase cleaves fatty acid hydroperoxides into aldehydes and oxoacids, and hydroperoxide dehydrase (also named hydroperoxide isomerase) catalyzes the formation of a-and 7-ketols. 

Hydroperoxide dehydrase activity was determined at 25 C by monitoring the decrease in absorbance at 234 nm [5] with 10 fjM of 13-hydroperoxide of linoleic acid as substrate. Hydroperoxide lyase activity was determined by a specific spectrophotometric assay [7], using 40 /iM of 13-hydroperoxide of linoleic acid as substrate. The solution of 13-hydroperoxide of... 

For partial purification of hydroperoxide dehydrase, greenmalt germs (1 g) were extracted in 5 ml of extraction buffer as described above. After centrifugation (30 min. at 10 OOOg), the supernatant was collected, passed through a 0.22 fim filter and diluted 2 times with water. On an anion exchange column (Mono Q HR 5/5, Pharmacia) equilibrated with buffer A (10 mM phoshate buffer, pH 7.5), 10 ml of extract were loaded. Sequential elution was performed on a FPLC (Pharmacia) at 0.5 ml/min using buffer B, buffer A and Buffer C (buffer B 1 M NaCl in buffer A, buffer C 0.1 % Triton X-100 in buffer A). 

Figure 1 Changes of hydroperoxide dehydrase activity during germination.

The optimum pH of the linoleate hydroperoxide dehydrase determined with the purified fraction was 6.6 with a broad activity range (Fig. 3). At pH 4 and pH 10, respectively 30% and 18 % of the maximum activity are still present. Despite the fact that hydroperoxide dehydrase has been shown to be moderately heat stable in crude extracts of commercial malts [6], the enzyme in the purified fraction shows a low temperature stability. A decline of 50 % in activity is observed after 3 hours at 4°C (data not shown). 

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