Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.     

Separation of enzymatic functions and variation of spin state of rice allene oxide synthase-1 by mutation of Phe-92 and Pro-430

Rice allene oxide synthase-1 mutants carrying F92L, P430A or F92L/P430A amino acid substitution mutations were constructed, recombinant mutant and wild type proteins were purified and their substrate preference, UV–vis spectra and heme iron spin state were characterized. The results show that the hydroperoxide lyase activities of F92L and F92L/P430A mutants prefer 13-hydroperoxy substrate to other hydroperoxydienoic acids or hydroperoxytrienoic acids. The Soret maximum was completely red-shifted in P430A and F92L/P430A mutants, but it was partially shifted in the F92L mutant. ESR spectral data showed that wild type, F92L and P430A mutants occupied high and low spin states, while the F92L/P430A mutant occupied only low spin state. The extent of the red shift of the Soret maximum increased as the population of low spin heme iron increased, suggesting that the spectral shift reflects the high to low transition of heme iron spin state in rice allene oxide synthase-1. Relative to wild type allene oxide synthase-1, the hydroperoxide lyase activities of F92L and F92L/P430A are less sensitive to inhibition by imidazole with (13S or 9S)-hydroperoxydienoic acid as substrate and more sensitive than wild type with (13S)-hydroperoxytrienoic acid as substrate. Our results suggest that hydroperoxydienoic acid is the preferred substrate for the hydroperoxide lyase activity and (13S)-hydroperoxytrienoic acid is the preferred substrate for allene oxide synthase activity of allene oxide synthase-1.     

The “heterolytic hydroperoxide lyase” is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10–60 s at 0 °C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1′-hydroxy-3′-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [¹⁸O₂]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [¹⁸O₂]13-HPOT retained two and one ¹⁸O atoms, respectively, whereas no ¹⁸O was incorporated from [¹⁸O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of fatty acid hydroperoxides into hemiacetals

The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.     

Traumatin- and Dinortraumatin-containing Galactolipids in Arabidopsis: THEIR FORMATION IN TISSUE-DISRUPTED LEAVES AS COUNTERPARTS OF GREEN LEAF VOLATILES*

Green leaf volatiles (GLVs) consisting of six-carbon aldehydes, alcohols, and their esters, are biosynthesized through the action of fatty acid hydroperoxide lyase (HPL), which uses fatty acid hydroperoxides as substrates. GLVs form immediately after disruption of plant leaf tissues by herbivore attacks and mechanical wounding and play a role in defense against attackers that attempt to invade through the wounds. The fates and the physiological significance of the counterparts of the HPL reaction, the 12/10-carbon oxoacids that are formed from 18/16-carbon fatty acid 13-/11-hydroperoxides, respectively, are largely unknown. In this study, we detected monogalactosyl diacylglycerols (MGDGs) containing the 12/10-carbon HPL products in disrupted leaf tissues of Arabidopsis, cabbage, tobacco, tomato, and common bean. They were identified as an MGDG containing 12-oxo-9-hydroxy-(E)-10-dodecenoic acid and 10-oxo-7-hydroxy-(E)-8-decenoic acid and an MGDG containing two 12-oxo-9-hydroxy-(E)-10-dodecenoic acids as their acyl groups. Analyses of Arabidopsis mutants lacking HPL indicated that these MGDGs were formed enzymatically through an active HPL reaction. Thus, our results suggested that in disrupted leaf tissues, MGDG-hydroperoxides were cleaved by HPL to form volatile six-carbon aldehydes and non-volatile 12/10-carbon aldehyde-containing galactolipids. Based on these results, we propose a novel oxylipin pathway that does not require the lipase reaction to form GLVs.     

The reaction mechanism of allene oxide synthase: Interplay of theoretical QM/MM calculations and experimental investigations

A combined theoretical and experimental study highlights the reaction mechanism of allene oxide synthase (AOS) and its possible link to hydroperoxide lyase (HPL) pathway. A previously published study (Lee et al., Nature 455 (2008) 363) has shown that the F137 residue is of central importance in differentiating between the AOS and HPL pathways after initial identical steps. In the experimental part of this study, we show that wild-type AOS from Arabidopsis or rice in fact produces both AOS and HPL products in a ratio of about 80:15, something that was found only in trace amounts before. Theoretical calculations successfully map the whole AOS pathway with 13(S)-hydroperoxy linolenic and linoleic acid as substrates. Subsequent calculations investigated the effects of in silico F137L mutation at the suggested diverging point of the two pathways. The results show that QM/MM calculations can reasonably reproduce three out of four experimentally available cases, and confirm that the pathways are energetically very close to each other, thus making a switch from one path to other plausible under different circumstances.     

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE*

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using ¹⁸O-labeled substrate and incubations in H₂¹⁸O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom.     

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.     

Detection of unprecedented allene oxide synthase member of CYP74B subfamily: CYP74B33 of carrot (Daucus carota)

Enzymes of CYP74 family widespread in higher plants control the metabolism of fatty acid hydroperoxides to numerous bioactive oxylipins. Hydroperoxide lyases (HPLs, synonym: hemiacetal synthases) of CYP74B subfamily belong to the most common CYP74 enzymes. HPLs isomerize the hydroperoxides to the short-lived hemiacetals, which are spontaneously decomposed to aldehydes and aldoacids. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) possessed the 13-HPL activity. Present work reports the cloning of the expressed CYP74B33 gene of carrot (Daucus carota L.) and studies of catalytic properties of the recombinant CYP74B33 protein. In contrast to all CYP74B proteins studied yet, CYP74B33 behaved differently in few respects. Firstly, the preferred substrates of CYP74B33 are 9-hydroperoxides. Secondly and most importantly, CYP74B33 exhibits the 9-allene oxide synthase (AOS) activity. For example, the 9(S)-hydroperoxide of linoleic acid (9-HPOD) underwent the conversion to α-ketol via the short-lived allene oxide. Uncommonly, the 9-HPOD conversion affords a minority of cis-10-oxo-11-phytoenoic acid, which is also produced by CYP74C but not the CYP74A AOSs. The similar product patterns were observed upon the incubations of CYP74B33 with 9(S)-hydroperoxide of α-linolenic acid. The enzyme possessed a mixed HPL, AOS, and the epoxyalcohol synthase activity toward the 13-hydroperoxides, but the total activity was much lower than toward 9-hydroperoxides. Thus, the obtained results show that CYP74B33 is an unprecedented 9-AOS within the CYP74B subfamily.     

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.     

C6-aldehyde formation by fatty acid hydroperoxide lyase in the brown alga Laminaria angustata

Some marine algae can form volatile aldehydes such as n-hexanal, hexenals, and nonenals. In higher plants it is well established that these short-chain aldehydes are formed from C18 fatty acids via actions of lipoxygenase and fatty acid hydroperoxide lyase, however, the biosynthetic pathway in marine algae has not been fully established yet. A brown alga, Laminaria angustata, forms relatively higher amounts of C6- and C9-aldehydes. When linoleic acid was added to a homogenate prepared from the fronds of this algae, formation of n-hexanal was observed. When glutathione peroxidase was added to the reaction mixture concomitant with glutathione, the formation of n-hexanal from linoleic acid was inhibited, and oxygenated fatty acids accumulated. By chemical analyses one of the major oxygenated fatty acids was shown to be (S)-13-hydroxy-(Z, E)-9, 11-octadecadienoic acid. Therefore, it is assumed that n-hexanal is formed from linoleic acid via a sequential action of lipoxygenase and fatty acid hydroperoxide lyase (HPL), by an almost similar pathway as the counterpart found in higher plants HPL partially purified from the fronds has a rather strict substrate specificity, and only 13-hydroperoxide of linoleic acid, and 15-hydroperoxide of arachidonic acid are the essentially suitable substrates for the enzyme. By surveying various species of marine algae including Phaeophyta, Rhodophyta and Chlorophyta it was shown that almost all the marine algae have HPL activity. Thus, a wide distribution of the enzyme is expected.     

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 lambda max 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.     

Formation of oxylipins by CYP74 enzymes

Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes. Products are hydroperoxy polyunsaturated fatty acids and metabolites derived there from collectively named oxylipins. They may either originate from chemical oxidation or are synthesized by the action of various enzymes, such as lipoxygenases. Cloning of many lipoxygenases and other key enzymes metabolizing oxylipins revealed new insights on oxylipin functions, new reactions and the first hints on enzyme mechanisms. These aspects are reviewed with respect to metabolism of fatty acid hydroperoxides by an atypical P450 subfamily: the CYP74. Up to now this protein family contains three different enzyme activities: (i) allene oxide synthase leading to the formation of unstable allene oxides which react to ketol and cyclopentenone fatty acids, (ii) hydroperoxide lyase producing hemiacetals decomposing to aldehydes and ω-oxo fatty acids and (iii) divinyl ether synthase which forms divinyl ethers. Signalling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among their numerous products.     

Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme

Five kinds of spacer arm attached chitosan hybrid hydrogels were tested for the possibility of being used as carriers for the immobilization of hydroperoxide lyase from amaranthus tricolor leaves. The 1,6-hexamethylenediamine attached chitosan-κ-carrageenan with biomimetic hydrophobic surface was proved to be the most suitable carrier. A maximum activity of 7.49 ± 0.19 U/g and a yield of 95% were obtained under optimized coupling condition. Meanwhile, the affinity between enzyme and substrates was not reduced after immobilization, as evidenced by the fact that the K_m value of hydroperoxide lyase decreased from 108.6 to 79.97 μM for 13-hydroperoxy-linoleic-acid and almost unchanged for 13-hydroperoxy-linolenic-acid. Furthermore, the thermal, operational and storage stabilities of HPL were significantly improved after immobilization. Using the immobilized enzyme as the catalyst, the yield of 2(E)-hexenal and hexanal reached 1374.8 ± 51.8 mg/L and 1987.9 ± 67.9 mg/L, respectively, and the amount of immobilized enzyme needed in the reaction mixture was much lower than its free counterpart.     

Effect of redox potential on the growth of Yarrowia lipolytica and the biosynthesis and activity of heterologous hydroperoxide lyase

The aim of this study was to investigate the effect of redox potential (Eh) on the growth of the yeast Yarrowia lipolytica in both oxidizing (Eh = +350 mV) and reducing (Eh = −150 mV) media and its effect on the expression and activity of hydroperoxide lyase (HPL). HPL activity was assayed in media with Eh values ranging from −250 to +720 mV. In order to change the Eh value of the media, reducing agents including dithiotreitol (1 g/L) and hydrogen (4%) as well as oxidants such as potassium ferricyanide (1 g/L) and oxygen (100%), were used. The experimental findings showed that oxidizing conditions, with Eh of +350 mV, were favorable for the growth of the yeast, whereas reducing conditions, with Eh values of −150 mV, resulted in a higher expression of HPL. In addition, the results showed that the enzymatic activity of the purified HPL was enhanced in the presence of 0.5 mM dithiotreitol but decreased with 1 mM potassium ferricyanide and bubbling O₂. However, HPL activity increased 1.5 times in the presence of 4% hydrogen with an Eh value of −170 mV.     

The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

Structural Characterization of Hydroperoxide Lyase in Dodecyl Maltoside by Using Circular Dichroism

Fatty acid hydroperoxide lyase (HPL) is a membrane protein, member of the lipoxygenase pathway, which holds a central role in plant defense. Green bell pepper fatty acid hydroperoxide lyase, overexpressed in Escherichia coli, was purified and solubilized in two different non ionic detergents, Triton X-100 and dodecyl maltoside (DM). DM is considered to be more useful compared to Triton X-100, as it allows characterization of the protein with spectroscopic techniques, for which Triton X-100 was inapplicable. Circular dichroism demonstrated that HPL’s secondary structure in DM consists of 13.53 % α-helix, 32.73 % β-sheet, 21.76 % turn and 31.13 % unordered.     

Biocatalysis with hydroperoxide lyase in extracts from Penicillium camemberti in neat organic solvent media

Abstract

Biocatalysis with hydroperoxide lyase (HPL) in extracts from Penicillium camemberti, in neat organic solvent media has been investigated. The effects of reaction conditions including organic solvent mixtures, initial water activity (a_w) and reaction temperature as well as the effect of the lyoprotectants, KCl and dextran 1 kDa, on HPL activity were studied. The addition of KCl to the enzymatic extract (70:1 protein, w/w) prior to lyophilization, enhanced HPL activity 6.53-fold. In contrast, the presence of dextran at a ratio of 8:1 decreased the enzymatic activity. Using hexane as the reaction medium, with an initial a_w of 0.1 and 0.5, the HPL specific activity was determined to be as 6.3 and 65.9 nmol converted 10-HPOD/mg protein/min, for the enzymatic extract without and with KCl present, respectively. Although HPL enzymatic extract with KCl showed a relatively low optimum reaction temperature (45°C) compared to 55°C without KCl, it exhibited a 2.51- and 2.78-fold higher thermal stability at 60 and 80°C, respectively. The kinetic results indicated that the highest HPL catalytic efficiency, V_max/K_m, of 6.58 × 10^?2 mL/mg protein/min, was obtained in the presence of KCl.     

Overexpression of hydroperoxide lyase,peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors

Plants produce short‐chain aldehydes and hydroxy fatty acids, which are important industrial materials, through the lipoxygenase pathway. Based on the information that lipoxygenase activity is up‐regulated in tobacco leaves upon infection with tobacco mosaic virus (TMV), we introduced a melon hydroperoxide lyase (CmHPL) gene, a tomato peroxygenase (SlPXG) gene and a potato epoxide hydrolase (StEH) into tobacco leaves using a TMV‐based viral vector system to afford aldehyde and hydroxy fatty acid production. Ten days after infiltration, tobacco leaves infiltrated with CmHPL displayed high enzyme activities of 9‐LOX and 9‐HPL, which could efficiently transform linoleic acid into C₉ aldehydes. Protein extracts prepared from 1 g of CmHPL‐infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of control vector‐infiltrated tobacco leaves (as an additional 9‐LOX source) produced 758 ± 75 μg total C₉ aldehydes in 30 min. The yield of C₉ aldehydes from linoleic acid was 60%. Besides, leaves infiltrated with SlPXG and StEH showed considerable enzyme activities of 9‐LOX/PXG and 9‐LOX/EH, respectively, enabling the production of 9,12,13‐trihydroxy‐10(E)‐octadecenoic acid from linoleic acid. Protein extracts prepared from 1 g of SlPXG‐infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of StEH‐infiltrated tobacco leaves produced 1738 ± 27 μg total 9,12,13‐trihydroxy‐10(E)‐octadecenoic acid isomers in 30 min. The yield of trihydroxyoctadecenoic acids from linoleic acid was 58%. C₉ aldehydes and trihydroxy fatty acids could likely be produced on a larger scale using this expression system with many advantages including easy handling, time‐saving and low production cost.     

Identification and functional analyses of two cDNAs that encode fatty acid 9-/13-hydroperoxide lyase (CYP74C) in rice

Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.