Hydroperoxide Substrate Research Articles

Characterization of thioredoxin-thioredoxin reductase system in Filifactor alocis.

Filifactor alocis is a newly appreciated member of the periodontal community with a strong periodontal disease correlation. Little is known about the survival mechanisms by which F.alocis copes with oxidative stress and establishes the infection within the local inflammatory microenvironment of the periodontal pocket. The aim of this study is to investigate if F.alocis putative peroxiredoxin/AhpC protein FA768 may constitute an alkyl hydroperoxide reductase system utilizing putative thioredoxin reductase protein FA608, and putative thioredoxin/glutaredoxin homolog FA1411/FA455. FA768, FA608, FA1411 and FA455 proteins from F.alocis were expressed and purified from Escherichia coli. Insulin and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reduction assays were performed to determine if purified FA1411 and FA455 proteins could be a substrate for FA608. The peroxidase activity of FA768 was examined by measuring its ability to reduce hydrogen peroxide (H2O2) with FA608 and FA1411/FA455 provided as the reducing systems. Further, the hydroperoxide substrate specificity of FA768 was analyzed by monitoring the NADPH oxidation in the presence of different peroxides, including H2O2, cumyl hydroperoxide (CHP), and tert-butyl hydroperoxide (t-BHP). In this study, we have demonstrated the existence of a functioning thioredoxin-dependent alkyl hydroperoxide system in F.alocis. This system is comprised of a thioredoxin reductase (FA608), a thioredoxin/glutaredoxin homolog (FA1411/FA455), and a typical 2-cysteine peroxiredoxin/AhpC (FA768). FA608, together with FA1411/FA455, can function as a thioredoxin reductase system to reduce insulin, DTNB, and FA768. FA455 is a glutaredoxin-like protein with thioredoxin functions in F.alocis. Both the FA768/FA608/FA1411 and FA768/FA608/FA455 reductase systems were NADPH-dependent and exhibited specificity for broad hydroperoxide substrates H2O2, CHP, and t-BHP. This is the first study of a thioredoxin dependent alkyl hydroperoxide system from a periodontal pathogen. This system is proposed to protect F.alocis against oxidative stress due to the likely absence of a catalase or an additional peroxiredoxin homolog.

Se site targeted-two circles antioxidant in GPx4–like catalytic peroxide degradation by polyphenols (−)-epigallocatechin gallate and genistein using SERS

A Se site targeted-two circles antioxidant of polyphenols EGCG and genistein in glutathione peroxidase 4 (GPx4)–like catalytic peroxide H2O2 and cumene hydroperoxide degradation was demonstrated by surface-enhanced Raman scattering (SERS). Se atom's active center is presenting a ‘low-oxidation’ and a ‘high-oxidation’ catalytic cycle. The former is oxidized to selenenic acid (SeO−) with a Raman bond at 619/ 610 cm−1 assigned to the νO − Se by the hydroperoxide substrate at 544/ 551 cm−1 assigned to ωHSeC decreased. Under oxidative stress, the enzyme shifted to ‘high-oxidation’ catalytic cycle, in which GPx4 shuttles between R-SeO− and R-SeOO− with a Raman intensity of bond at 840/ 860 cm−1 assigned to νOSe. EGCG could act as a reducing agent both in H2O2 and Cu-OOH degradation, while, genistein can only reduce Cu-OOH, because it binds more readily to the selenium site in GPx4 than EGCG with a closer proximity, therefore may affect its simultaneous binding to coenzymes.

Biosynthesis of natural aroma compounds using recombinant whole-cell tomato hydroperoxide lyase biocatalyst

Green leaf volatiles impart characteristic aroma and flavour to a variety of natural foods due to their inherent grassy note contributed by aldehydes. Hydroperoxide lyase (HPL) is an enzyme that helps in the cleavage of fatty acid hydroperoxides to short-chain aldehydes and ω-oxo-acids. A tomato hydroperoxide lyase gene was successfully expressed in E. coli BL21 (DE3) cells and used in the subsequent production of (Z)-3-hexenal. Biochemical characterization of the HPL activity exhibited by these whole cells enabled the development of a suitable one-pot reaction process for conversion of the hydroperoxide substrate to the corresponding aldehyde, (Z)-3-hexenal, and finally to (Z)-3-hexenol, a high-value flavour and fragrance ingredient.

Protein homology modeling‐informed active site characterization of a bacterial prostaglandin synthase ortholog from Nitrosomonas europaea

An open reading frame in the genome of Nitrosomonas europaea has been annotated as a prostaglandin H synthase (PGHS). We have expressed the protein, which demonstrates peroxidase activity with both hydrogen peroxide and lipid hydroperoxide substrates. Dioxygenase activity was not observed. In this study, we generate models of the N. europaea peroxidase to understand the structural explanation for its observed function. Using SWISS-MODEL, protein homology models were generated against PGHS, peroxidase, and dioxygenase templates. Using AutoDock Vina, the substrate 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid was inserted into the active site of each model to identify potential amino acids critical for activity. A conserved di-tyrosyl structure was observed in several unique models generated against redox enzyme templates. Amino acids in the active site predicted in the protein homology models were modified by site-directed mutagenesis. Specifically, the conserved tyrosyl structure was mutated to elucidate the importance of the structure in addition to the individual residues. Wild-type and mutant enzymes were expressed in BL21 cells and purified by Ni-NTA and Sephacryl S-200 chromatography. Kinetic assays using hydrogen peroxide and lipid hydroperoxide substrates are in progress to compare the mutant and wild-type enzyme activity. Identifying critical residues will contribute to our understanding of the evolutionary development of mammalian prostaglandin synthase.

A dual attack on the peroxide bond. The common principle of peroxidatic cysteine or selenocysteine residues

The (seleno)cysteine residues in some protein families react with hydroperoxides with rate constants far beyond those of fully dissociated low molecular weight thiol or selenol compounds. In case of the glutathione peroxidases, we could demonstrate that high rate constants are achieved by a proton transfer from the chalcogenol to a residue of the active site [Orian et al. Free Radic. Biol. Med. 87 (2015)]. We extended this study to three more protein families (OxyR, GAPDH and Prx). According to DFT calculations, a proton transfer from the active site chalcogenol to a residue within the active site is a prerequisite for both, creating a chalcogenolate that attacks one oxygen of the hydroperoxide substrate and combining the delocalized proton with the remaining OH or OR, respectively, to create an ideal leaving group. The “parking postions” of the delocalized proton differ between the protein families. It is the ring nitrogen of tryptophan in GPx, a histidine in GAPDH and OxyR and a threonine in Prx. The basic principle, however, is common to all four families of proteins. We, thus, conclude that the principle outlined in this investigation offers a convincing explanation for how a cysteine residue can become peroxidatic.

Radical-Pair Formation in Hydrocarbon (Aut)Oxidation.

The reaction profiles for the uni- and bimolecular decomposition of benzyl hydroperoxide have been studied in the context of initiation reactions for the (aut)oxidation of hydrocarbons. The unimolecular dissociation of benzyl hydroperoxide was found to proceed through the formation of a hydrogen-bonded radical-pair minimum located +181 kJ mol-1 above the hydroperoxide substrate and around 15 kJ mol-1 below the separated radical products. The reaction of toluene with benzyl hydroperoxide proceeds such that O-O bond homolysis is coupled with a C-H bond abstraction event in a single kinetic step. The enthalpic barrier of this molecule-induced radical formation (MIRF) process is significantly lower than that of the unimolecular O-O bond cleavage. The same type of reaction is also possible in the self-reaction between two benzyl hydroperoxide molecules forming benzyloxyl and hydroxyl radical pairs along with benzaldehyde and water as co-products. In the product complexes formed in these MIRF reactions, both radicals connect to a centrally placed water molecule through hydrogen-bonding interactions.

Comprehensive analysis of antioxidant mechanisms in Arabidopsis glutathione peroxidase-like mutants under salt- and osmotic stress reveals organ-specific significance of the AtGPXL’s activities

Plant glutathione peroxidases contain cysteine in their active site instead of selenocysteine, and most of them use the thioredoxin (TRX) system more efficiently than the glutathione (GSH) system during the reduction of H2O2 and lipid peroxides. Recently, the more precise glutathione peroxidase-like (GPXL) name was adopted for the eight Arabidopsis thaliana isoenzymes. In this paper we have compared the effect of osmotic and salt stresses on the 6-week-old T-DNA insertion mutants (Atgpxl1-8) grown hydroponically. The glutathione peroxidase (GPOX) activity measured with cumene hydroperoxide substrate in the wild type Arabidopsis shoots and roots was 2–5 times higher than the thioredoxin peroxidase (TPOX) activity. Mutation in one of the eight AtGPXLs resulted in decreased TPOX activity in untreated shoots and, in contrary to the wild type, this activity did not increase under stress, verifying the connection with TRX system. The level of reduced ascorbate significantly altered in shoots and the amount of GSH in roots under both control conditions and after 2 days of stress treatments. While positive correlations were found between GSH and TPOX activity in the wild type shoots and roots, the connection between the AtGPXLs and the GSH pool was stronger in roots than in shoots. Nevertheless, the TPOX activity increased in Atgpxls roots when the GSH content decreased, indicating the relationship between the GSH and TRX systems. The AtGPXL expression in mutant plants showed that some isoenzymes are regulated jointly. Furthermore, while AtGPXLs were generally down-regulated, several stress-inducible transcription factor genes were up-regulated, especially after applying osmotic stress.

Catalase-Related Allene Oxide Synthase, on a Biosynthetic Route to Fatty Acid Cyclopentenones: Expression and Assay of the Enzyme and Preparation of the 8R-HPETE Substrate.

Catalase-related allene oxide synthase (cAOS) is a hemoprotein that converts a specific fatty acid hydroperoxide to an unstable allene oxide intermediate at turnover rates in the order of 1000 per second. Fatty acid allene oxides are intermediates in the formation of cyclopentenone or hydrolytic products in marine systems, most notably the prostanoid-related clavulones. Although the key catalytic amino acid residues around the active site of cAOS are the same as in true catalases, cAOS does not react with hydrogen peroxide. cAOS occurs exclusively as the N-terminal domain of a naturally occurring fusion protein with a C-terminal lipoxygenase (LOX) domain that supplies the hydroperoxide substrate. In marine invertebrates, an 8R-LOX domain converts arachidonic acid to 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) and the cAOS domain forms an 8,9-epoxy allene oxide. The fusion protein from the sea whip octocoral Plexaura homomalla is the prototypical model with crystal structures of the individual domains. The cAOS (43kDa) expresses exceptionally well in Escherichia coli, with yields of up to 100mg/L. This article describes in detail expression and assay of the P. homomalla cAOS and two methods for the preparation of its 8R-HPETE substrate. Another article in this volume focuses on the P. homomalla 8R-LOX (Gilbert, Neau, & Newcomer, 2018).

A fungal catalase reacts selectively with the 13S fatty acid hydroperoxide products of the adjacent lipoxygenase gene and exhibits 13S-hydroperoxide-dependent peroxidase activity

The genome of the fungal plant pathogen Fusarium graminearum harbors six catalases, one of which has the sequence characteristics of a fatty acid peroxide-metabolizing catalase. We cloned and expressed this hemoprotein (designated as Fg-cat) along with its immediate neighbor, a 13S-lipoxygenase (cf. Brodhun et al., PloS One, e64919, 2013) that we considered might supply a fatty acid hydroperoxide substrate. Indeed, Fg-cat reacts abruptly with the 13S-hydroperoxide of linoleic acid (13S-HPODE) with an initial rate of 700–1300s−1. By comparison there was no reaction with 9R- or 9S-HPODEs and extremely weak reaction with 13R-HPODE (~0.5% of the rate with 13S-HPODE). Although we considered Fg-cat as a candidate for the allene oxide synthase of the jasmonate pathway in fungi, the main product formed from 13S-HPODE was identified by UV, MS, and NMR as 9-oxo-10E-12,13-cis-epoxy-octadecenoic acid (with no traces of AOS activity). The corresponding analog is formed from the 13S-hydroperoxide of α-linolenic acid along with novel diepoxy-ketones and two C13 aldehyde derivatives, the reaction mechanisms of which are proposed. In a peroxidase assay monitoring the oxidation of ABTS, Fg-cat exhibited robust activity (kcat 550s−1) using the 13S-hydroperoxy-C18 fatty acids as the oxidizing co-substrate. There was no detectable peroxidase activity using the corresponding 9S-hydroperoxides, nor with t-butyl hydroperoxide, and very weak activity with H2O2 or cumene hydroperoxide at micromolar concentrations of Fg-cat. Fg-cat and the associated lipoxygenase gene are present together in fungal genera Fusarium, Metarhizium and Fonsecaea and appear to constitute a partnership for oxidations in fungal metabolism or defense.

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 (18)O-labeled substrate and incubations in H2(18)O, 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.

Dissecting peroxiredoxin catalysis: separating binding, peroxidation, and resolution for a bacterial AhpC.

Peroxiredoxins make up a ubiquitous family of cysteine-dependent peroxidases that reduce hydroperoxide or peroxynitrite substrates through formation of a cysteine sulfenic acid (R-SOH) at the active site. In the 2-Cys peroxiredoxins, a second (resolving) cysteine reacts with the sulfenic acid to form a disulfide bond. For all peroxiredoxins, structural rearrangements in the vicinity of the active site cysteine(s) are necessary to allow disulfide bond formation and subsequent reductive recycling. In this study, we evaluated the rate constants for individual steps in the catalytic cycle of Salmonella typhimurium AhpC. Conserved Trp residues situated close to both peroxidatic and resolving cysteines in AhpC give rise to large changes in fluorescence during the catalytic cycle. For recycling, AhpF very efficiently reduces the AhpC disulfide, with a single discernible step and a rate constant of 2.3 × 10(7) M(-1) s(-1). Peroxide reduction was more complex and could be modeled as three steps, beginning with a reversible binding of H2O2 to the enzyme (k1 = 1.36 × 10(8) M(-1) s(-1), and k-1 = 53 s(-1)), followed by rapid sulfenic acid generation (620 s(-1)) and then rate-limiting disulfide bond formation (75 s(-1)). Using bulkier hydroperoxide substrates with higher Km values, we found that different efficiencies (kcat/Km) for turnover of AhpC with these substrates are primarily caused by their slower rates of binding. Our findings indicate that this bacterial peroxiredoxin exhibits rates for both reducing and oxidizing parts of the catalytic cycle that are among the fastest observed so far for this diverse family of enzymes.

Pseudoperoxidase investigations of hydroperoxides and inhibitors with human lipoxygenases

Understanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result.

Molecular dynamics simulations of cyclodextrin–cumene hydroperoxide complexes in water

It has well been known that glutathione peroxidase (GPX) mimics based on cyclodextrins (CDs) have great selectivity to hydroperoxide substrates and the preferred hydroperoxide is the aromatic cumene hydroperoxide (CuOOH). The reduction of CuOOH often proceeds much faster than reduction of the more hydrophilic hydroperoxides. The purpose of this study is to provide theoretical evidence of this substrate specificity mechanism. In this contribution, we report our investigation on the intermolecular interaction and modeling calculations on the complexes of three cyclodextrins, viz. α-, β-, and γ-CD, with CuOOH by means of computational molecular dynamics (MD) simulations. The free energy profile along the ordering parameter, association constant, and the corresponding association free energy, as well as the most important interactions which contribute to their stability were studied in detail. The results show that stable inclusion complexes only form when both the host and guest molecules experience a significant decrease in the complexing potential. Among the three CDs, β-CD exhibits the highest propensity to associate with CuOOH. Ranking for binding CuOOH, viz. β-CD>γ-CD>α-CD.

Destroy and Exploit: Catalyzed Removal of Hydroperoxides from the Endoplasmic Reticulum

Peroxidases are enzymes that reduce hydroperoxide substrates. In many cases, hydroperoxide reduction is coupled to the formation of a disulfide bond, which is transferred onto specific acceptor molecules, the so-called reducing substrates. As such, peroxidases control the spatiotemporal distribution of diffusible second messengers such as hydrogen peroxide (H2O2) and generate new disulfides. Members of two families of peroxidases, peroxiredoxins (Prxs) and glutathione peroxidases (GPxs), reside in different subcellular compartments or are secreted from cells. This review discusses the properties and physiological roles of PrxIV, GPx7, and GPx8 in the endoplasmic reticulum (ER) of higher eukaryotic cells where H2O2 and—possibly—lipid hydroperoxides are regularly produced. Different peroxide sources and reducing substrates for ER peroxidases are critically evaluated. Peroxidase-catalyzed detoxification of hydroperoxides coupled to the productive use of disulfides, for instance, in the ER-associated process of oxidative protein folding, appears to emerge as a common theme. Nonetheless, in vitro and in vivo studies have demonstrated that individual peroxidases serve specific, nonoverlapping roles in ER physiology.

Measurement of peroxiredoxin activity.

Peroxiredoxins are cysteine-dependent peroxidases that react with hydrogen peroxide, larger hydroperoxide substrates, and peroxynitrite. Protocols are provided to measure Prx activity with peroxide by (1) a coupled reaction with NADPH, thioredoxin reductase, and thioredoxin, (2) the direct monitoring of thioredoxin oxidation, (3) competition with horseradish peroxidase, and (4) peroxide consumption using the FOX assay.

Broad specificity AhpC-like peroxiredoxin and its thioredoxin reductant in the sparse antioxidant defense system of Treponema pallidum

Little is known about the mechanisms by which Treponema pallidum (Tp), the causative agent of syphilis, copes with oxidative stress as it establishes persistent infection within its obligate human host. The Tp genomic sequence indicates that the bacterium's antioxidant defenses do not include glutathione and are limited to just a few proteins, with only one, TP0509, offering direct defense against peroxides. Although this Tp peroxiredoxin (Prx) closely resembles AhpC-like Prxs, Tp lacks AhpF, the typical reductant for such enzymes. Functionally, TpAhpC resembles largely eukaryotic, nonAhpC typical 2-Cys Prx proteins in using thioredoxin (Trx, TP0919) as an efficient electron donor and exhibiting broad specificity toward hydroperoxide substrates. Unlike many of the eukaryotic Prxs, however, TpAhpC is relatively resistant to inactivation during turnover with hydroperoxide substrates. As is often observed in typical 2-Cys Prxs, TpAhpC undergoes redox-sensitive oligomer formation. Quantitative immunoblotting revealed that TpTrx and TpAhpC are present at very high levels (over 100 and 300 microM, respectively) in treponemes infecting rabbit testes; their redox potentials, at -242 +/- 1 and -192 +/- 2 mV, respectively, are consistent with the role of TpTrx as the cellular reductant of TpAhpC. Transcriptional analysis of select antioxidant genes confirmed the presence of high mRNA levels for ahpC and trx which diminish greatly when spirochetes replicate under in vitro growth conditions. Thus, T. pallidum has evolved an extraordinarily robust, broad-spectrum AhpC as its sole mechanism for peroxide defense to combat this significant threat to treponemal growth and survival during infection.

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation (Hamberg, M. 1983. Autoxidation of linoleic acid: Isolation and structure of four dihydroxy octadecadienoic acids. Biochim. Biophys. Acta 752: 353-356) and in incubations with the Anabaena enzyme (Lang, I., C. Göbel, A. Porzel, I. Heilmann, and I. Feussner. 2008. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120. Biochem. J. 410: 347-357). We also prepared an equivalent epoxide from 13S-hydroperoxylinoleate using a "biomimetic" chemical method originally described for LTA(4) synthesis and showed that like LTA(4), the C18.2 epoxide conjugates readily with glutathione, a potential metabolic fate in vivo. We compare and contrast the mechanisms of LTA-type allylic epoxide synthesis by lipoxygenase, catalase/peroxidase, and chemical transformations. These findings provide new insights into the reactions of linoleic acid hydroperoxides and extend the known range of catalytic activities of catalase-related hemoproteins.

Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes

The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic pi-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.

Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous peroxidases that are involved in peroxide scavenging and/or the regulation of peroxide signaling in eukaryotes. Despite their prevalence, very few Prxs have been reliably characterized in terms of their substrate specificity profile and redox potential even though these values are important for gaining insight into physiological function. Here, we present such studies focusing on Salmonella typhimurium alkyl hydroperoxide reductase C component (StAhpC), an enzyme that has proven to be an excellent prototype of this largest and most widespread class of Prxs that includes mammalian Prx I-Prx IV. The catalytic efficiencies of StAhpC (k(cat)/K(m)) are >10(7) M(-1).s(-1) for inorganic and primary hydroperoxide substrates and approximately 100-fold less for tertiary hydroperoxides, with the difference being exclusively caused by changes in K(m). The oxidative inactivation of AhpC through reaction with a second molecule of peroxide shows parallel substrate specificity. The midpoint reduction potential of StAhpC is determined to be -178 +/- 0.4 mV, a value much higher than most other thiol-based redox proteins. The relevance of these results for our understanding of Prx and the physiological role of StAhpC is discussed.