Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation.

Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme. The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin. EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding. Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation. Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature. Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.

Fatty acid hydroperoxide lyase: a plant cytochrome p450 enzyme involved in wound healing and pest resistance.

Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The first step in the lipoxygenase pathway is the reaction of linoleic or linolenic acids with molecular oxygen, catalyzed by the enzyme lipoxygenase. The hydroperoxy fatty acids thus formed are highly reactive and dangerous for the plant and therefore further metabolized by other enzymes such as allene oxide synthase, hydroperoxide lyase, peroxygenase, or divinyl ether synthase. Recently, these enzymes have been characterized as a special class of cytochrome P450 enzymes. Hydroperoxide lyases cleave the lipoxygenase products, resulting in the formation of omega-oxo acids and volatile C6- and C9-aldehydes and -alcohols. These compounds are major contributors to the characteristic "fresh green" odor of fruit and vegetables. They are widely used as food flavors, for example, to restore the freshness of food after sterilization processes. The low abundance of these compounds in nature and the high demand make it necessary to synthesize them on a large scale. Lipoxygenase and hydroperoxide lyase are suitable biocatalysts for the production of "natural" food flavors. In contrast to lipoxygenase, which has been extensively studied, little is yet known about hydroperoxide lyase. Hydroperoxide lyases from different organisms have been isolated, and a few genes have been published lately. However, the structure and reaction mechanism of this enzyme are still unclear. The identification of this enzyme as a cytochrome P450 sheds new light on its structure and possible reaction mechanism, whereas recombinant expression brings a biocatalytic application into sight.

Oxygenation of (3Z)-alkenals to 4-hydroxy-(2E)-alkenals in plant extracts: a nonenzymatic process.

There is large interest in 4-hydroxy-(2E)-alkenals because of their cytotoxicity in mammals. However, the biosynthetic pathway for these compounds has not been elucidated yet. In plants, 4-hydroxy-(2E)-alkenals were supposed to be derived by the subsequent actions of lipoxygenase and a peroxygenase on (3Z)-alkenals. The presence of 9-hydroxy-12-oxo-(10E)-dodecenoic acid (9-hydroxy-traumatin) in incubations of 12-oxo-(9Z)-dodecenoic acid (traumatin) in the absence of lipoxygenase or peroxygenase, has prompted us to reinvestigate its mode of formation. We show here that in vitro 9-hydroxy-traumatin, 4-hydroxy-(2E)-hexenal and 4-hydroxy-(2E)-nonenal, are formed in a nonenzymatic process. Furthermore, a novel product derived from traumatin was observed and identified as 11-hydroxy-12-oxo-(9Z)-dodecenoic acid. The results obtained here strongly suggest that the 4-hydroxy-(2E)-alkenals, observed in crude extracts of plants, are mainly due to autoxidation of (3Z)-hexenal, (3Z)-nonenal and traumatin. This may have implications for the in vivo existence and previously proposed physiological significance of these products in plants.

Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics.

Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases.

Formation of a new class of oxylipins from N-acyl(ethanol)amines by the lipoxygenase pathway.

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both alpha-N-linolenoylethanolamine and gamma-N-linolenoylethanolamine (18:3), as well as alpha-N-linolenoylamine and gamma-N-linolenoylamine were converted into their (13S)-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of alpha-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z)-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)-hydroperoxy-alpha-N-linolenoylethanolamine and the (13S)-hydroperoxide of linoleic acid and its ethanolamine derivative. alpha-Ketols and gamma-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S)-hydroperoxy-alpha-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.

Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase.

Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been reported to contain a hydroperoxide lyase specific for 13-hydroperoxy linoleic and linolenic acid only. However, in addition to 13-hydroperoxide lyase activity we found substantial 9-hydroperoxide lyase activity in alfalfa seedlings as well. The specific activity for 9-hydroperoxy fatty acids was about 50% of the activity for the 13-isomers. Furthermore, alfalfa seedlings contain a 3Z:2E-enal isomerase that converts the 3Z-enal products to their 2E-enal isoforms.

Pore formation by nisin involves translocation of its C-terminal part across the membrane.

Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.

Iron release from human monocytes after erythrophagocytosis in vitro: an investigation in normal subjects and hereditary hemochromatosis patients.

This study investigated the release of erythrocyte-derived iron from purified human monocytes obtained from healthy volunteers and hereditary hemochromatosis (HH) patients. After erythrophagocytosis of 59Fe-labeled erythrocytes, a complete transfer of iron from hemoglobin (Hb) to ferritin was observed within 24 hours in both control and HH monocytes. The iron was released from the monocytes in the form of ferritin, Hb, and as nonprotein bound low molecular weight iron (LMW-Fe). During the initial rapid phase (<1.5 hours), iron release mostly consisted of Hb and LMW-Fe, while in the later phase (>1.5 hours), it was composed of ferritin and LMW-Fe. The kinetics of iron release were identical for HH monocytes. A high percentage of the total amount of iron was released as Hb both by viable normal and HH monocytes, suggesting that iron release as Hb is a physiologic process, which may occur whenever the erythrocyte-processing capacity of macrophages is exceeded. Most remarkably, HH monocytes released twice as much iron in a LMW form as control cells. Iron released in the form of LMW-Fe readily binds to plasma transferrin and may contribute to the high transferrin saturation and the occurrence of circulating nontransferrin-bound iron observed in HH patients.