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Kinetic studies have indicated that imidazole is a competitive inhibitor of rice allene oxide synthase-1 with Ki of 34.9, 14.3, 40.3, and 23.3 mM for 13( S)- HPODE, 13( S)- HPOTE, 9( S)- HPODE, and 9( S)- HPOTE as substrates, respectively. Imidazole-free rice allene oxide synthase-1 has a Soret absorbance maximum at 393 nm and occupied high-spin ( g = 6.0) and low-spin ( g = 2.39, 2.24, 1.93) states in the ratio 1:10, as detected by EPR spectroscopy, while in the presence of imidazole the occupancy of the low-spin state increased to 100%. The results support the hypothesis that imidazole competes with the substrate of allene oxide synthase-1 for binding to the sixth position of heme iron( III) and that binding of imidazole stabilizes the low-spin state. The role of detergent binding and implications for the mechanism by which allene oxide synthase-1 cleaves the RO-OH bond are discussed. [ABSTRACT FROM AUTHOR]

Salinity stress represents a global constraint for rice, the most important staple food worldwide. Therefore the role of the central stress signal jasmonate for the salt response was analysed in rice comparing the responses to salt stress for two jasmonic acid (JA) biosynthesis rice mutants (cpm2 and hebiba) impaired in the function of ALLENE OXIDE CYCLASE (AOC) and their wild type. The aoc mutants were less sensitive to salt stress. Interestingly, both mutants accumulated smaller amounts of Na+ ions in their leaves, and showed better scavenging of reactive oxygen species (ROS) under salt stress. Leaves of the wild type and JA mutants accumulated similar levels of abscisic acid (ABA) under stress conditions, and the levels of JA and its amino acid conjugate, JA-isoleucine (JA-Ile), showed only subtle alterations in the wild type. In contrast, the wild type responded to salt stress by strong induction of the JA precursor 12-oxophytodienoic acid (OPDA), which was not observed in the mutants. Transcript levels of representative salinityinduced genes were induced less in the JA mutants. The absence of 12-OPDA in the mutants correlated not only with a generally increased ROS-scavenging activity, but also with the higher activity of specific enzymes in the antioxidative pathway, such as glutathione S-transferase, and fewer symptoms of damage as, for example, indicated by lower levels of malondialdehyde. The data are interpreted in a model where the absence of OPDA enhanced the antioxidative power in mutant leaves. [ABSTRACT FROM AUTHOR]

Allene oxide synthase (AOS), which is a cytochrome P450 (CYP74A), catalyses the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived hydroperoxides of free fatty acids. The full-length cDNA of anAOS-like gene was cloned fromBrassica oleraceausing rapid amplification of cDNA ends and was designated asBoAOS. TheBoAOSexpression level was higher in alabastrums and flowers than in other tissues of cabbage, as determined by real-time reverse transcription polymerase chain reaction (RT-PCR). To characterize theBoAOSgene,Arabidopsiswas transformed viaAgrobacterium tumefacienswith a vector to express the gene under the control of a strong constitutive promoter, CaMV35S (Cauliflower Mosaic Virus). Based on analyses of tolerance to drought stress, compared with control plants, the overexpression ofBoAOSin transgenic plants increased the endogenous jasmonic acid level and conferred higher tolerance to drought stress. Therefore, we suggest thatBoAOSmay be a suitable candidate gene to produce transgenic plants with tolerance to drought stress. [ABSTRACT FROM AUTHOR]

Marine ecosystems are sensitive to elevated seawater temperature, with stony corals serving as model organisms for temperature-imposed declines in population viability and diversity. Several stress markers, including heat shock proteins, have been used for the detection and prediction of stress responses in stony corals. However, the stress indicators in soft corals remain elusive. In higher animals and plants, oxylipins synthesized by fatty acid di- and monooxygenases contribute to stress-induced signaling; however, the role of eicosanoid pathways in corals remains unclear. The eicosanoid gene specific to corals encodes for a natural fusion protein of allene oxide synthase and lipoxygenase ( AOS- LOX). In this work, using the easily cultivated soft coral Capnella imbricata as the stress response model, we monitored the expression of the AOS-LOX and the formation of arachidonic acid metabolites in response to an acute rise in water temperature. Gene expression profiles of two 70 kDa heat shock proteins ( Hsps: Hsp70 and Grp78) were used as a positive control for the stress response. In comparison with normal seawater temperature (23 °C), AOS- LOXa and Hsps were all up-regulated after modest (28 °C) and severe (31 °C) temperature elevation. While the up-regulation of AOS- LOXa and Grp78 was more sensitive to moderate temperature changes, Hsp70s were more responsive to severe heat shock. Concurrently, endogenous and exogenous AOS-LOXa-derived eicosanoids were up-regulated. Thus, together with the up-regulation of AOS- LOX by other abiotic and biotic stress stimuli, these data implicate AOS-LOX as part of the general stress response pathway in corals. [ABSTRACT FROM AUTHOR]

Allene oxide synthase-1 from Oryza sativa (OsAOS1) localizes to the chloroplast, but lacks a putative chloroplast targeting sequence typically found in dicot AOS. Here, kinetic parameters and the oligomerization state/subunit composition of OsAOS1 were characterized in vitro in the absence or presence of detergent micelles. The catalytic efficiency ( k/ K) of OsAOS1 reached a maximum near the critical micelle concentration for polyoxyethylene 10 tridecyl ether. Native gel analysis showed that OsAOS1 exists as a multimer in the absence of detergent micelles. The multimeric form of OsAOS1 was stably cross-linked in the absence of detergents, while only monomeric OsAOS1 was detected in the presence of detergent micelles. Gel filtration analysis indicated that the oligomeric state of OsAOS1 depends strongly on the detergents and that the monomer becomes the predominant form in the presence of detergent micelles. These data suggest that the detergent-dependent oligomeric state of OsAOS1 is an important factor for the regulation of its catalytic efficiency. [ABSTRACT FROM AUTHOR]

True catalases efficiently breakdown hydrogen peroxide, whereas the catalase-related enzyme allene oxide synthase (cAOS) is completely unreactive and instead metabolizes a fatty acid hydroperoxide. In cAOS a Thr residue adjacent to the distal His restrains reaction with H 2 O 2 (Tosha et al. (2006) J. Biol. Chem. 281:12610; De Luna et al. (2013) J. Phys. Chem. B 117: 14635) and its mutation to the consensus Val of true catalases permits the interaction. Here we investigated the effects of the reciprocal experiment in which the Val74 of human catalase is mutated to Thr, Ser, Met, Pro, or Ala. The Val74Thr substitution decreased catalatic activity by 3.5-fold and peroxidatic activity by 3-fold. Substitution with Ser had similar negative effects (5- and 3-fold decreases). Met decreased catalatic activity 2-fold and eliminated peroxidatic activity altogether, whereas the Val74Ala substitution was well tolerated. (The Val74Pro protein lacked heme). We conclude that the conserved Val74 of true catalases helps optimize catalysis. There are rare substitutions of Val74 with Ala, Met, or Pro, but not with Ser of Thr, possibly due their hydrogen bonding affecting the conformation of His75, the essential distal heme residue for activity in catalases. [ABSTRACT FROM AUTHOR]

We investigated the effect of the SHAM treatment of tea plants on their induced defense on a tea geometrid (TG), Ectropis obliqua Prout. Treatment of tea leaves with SHAM reduced the performance of TG and TG-elicited level of the lipoxygenase gene CsiLOX1 and the putative allene oxide synthase gene CsiAOS1. The release of wound-induced green leaf volatiles (GLVs) and the expression of the hydroperoxide lyase (HPL) gene CsiHPL1 were also reduced by SHAM treatment. The negative effect of SHAM dramatically reduced the total hebivore-induced plant volatiles (HIPVs) and the attractiveness to the parasitoid wasp Apanteles sp. These results indicated that SHAM may negatively mediate tea defense response against TG by modulating the wound-induced emission of GLVs, the expression of genes involved in oxylipin pathway, and the emission of other HIPV compounds that mediate direct and indirect defenses. [ABSTRACT FROM AUTHOR]

QM cluster and QM/MM protein models have been employed to understand aspects of the reaction mechanism of plant allene oxide synthase (pAOS). In this study we have investigated two reaction mechanisms for pAOS. The standard pAOS mechanism was contrasted with an alternative involving an additional active site molecule which has been shown to facilitate proton coupled electron transfer (PCET) in related systems. Firstly, we found that the results from QM/MM protein model are comparable with those from the QM cluster model, presumably due to the large active site used. Furthermore, the results from the QM cluster model show that the FeIII and FeIV pathways for the standard mechanism have similar energetic and structural properties, indicating that the reaction mechanism may well proceed via both pathways. However, while the PCET process is facilitated by an additional active site bound water in other related families, in pAOS it is not, suggesting this type of process is not general to all closely related family members. [ABSTRACT FROM AUTHOR]

Soybean ( Glycine max L.) displays considerable variation in its resistance to insects. Characterization and application of insect resistance genes in soybean germplasm may be a sustainable and economical approach to soybean production. In this study, we demonstrated that the gene encoding allene oxide synthase ( GmAOS1) was associated with soybean defense against the common cutworm (CCW, Spodoptera litura Fabricius) attack through gene expression analysis and association analysis. The expression of GmAOS1 in stems and leaves of resistant accessions was higher than that of susceptible accessions in the undamaged soybean plant. After CCW feeding, the transcript levels of GmAOS1 increased and reached their maximal values sooner in the leaves of resistant accession than they did in susceptible accession. The genomic sequence of GmAOS1 was re-sequenced and aligned in 184 soybean accessions. Forty-three SNPs/Indels were found in its promoter region and only one single nucleotide polymorphism was found in its coding region. Association analysis showed that GmAOS1 promoter polymorphisms were significantly associated with three soybean defense indexes. GmAOS1_19 was the most resistant allele associated with soybean resistance to CCW. Two elements (TATCCAT/C motif and Box II-like sequence) located at genomic regions upstream of GmAOS1_21 were significantly associated with compensatory regrowth of soybean plants. Gene expression analysis indicated that the accessions containing different GmAOS1 promoters and with different CCW resistance grades showed different expression levels of GmAOS1. Our data indicate that variation in GmAOS1 promoter is associated with soybean defense against CCW attack. [ABSTRACT FROM AUTHOR]

Oxylipins are signaling molecules formed enzymatically or spontaneously from unsaturated fatty acids in all aerobic organisms. Oxylipins regulate growth, development, and responses to environmental stimuli of organisms. The oxylipin biosynthesis pathway in plants includes a few parallel branches named after first enzyme of the corresponding branch as allene oxide synthase, hydroperoxide lyase, divinyl ether synthase, peroxygenase, epoxy alcohol synthase, and others in which various biologically active metabolites are produced. Oxylipins can be formed non-enzymatically as a result of oxygenation of fatty acids by free radicals and reactive oxygen species. Spontaneously formed oxylipins are called phytoprostanes. The role of oxylipins in biotic stress responses has been described in many published works. The role of oxylipins in plant adaptation to abiotic stress conditions is less studied; there is also obvious lack of available data compilation and analysis in this area of research. In this work we analyze data on oxylipins functions in plant adaptation to abiotic stress conditions, such as wounding, suboptimal light and temperature, dehydration and osmotic stress, and effects of ozone and heavy metals. Modern research articles elucidating the molecular mechanisms of oxylipins action by the methods of biochemistry, molecular biology, and genetics are reviewed here. Data on the role of oxylipins in stress signal transduction, stress-inducible gene expression regulation, and interaction of these metabolites with other signal transduction pathways in cells are described. In this review the general oxylipin-mediated mechanisms that help plants to adjust to a broad spectrum of stress factors are considered, followed by analysis of more specific responses regulated by oxylipins only under certain stress conditions. New approaches to improvement of plant resistance to abiotic stresses based on the induction of oxylipin-mediated processes are discussed. [ABSTRACT FROM AUTHOR]

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates ( JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13- AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato ( Solanum tuberosum), there are two putative St13- AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13- AOS enzymes. Indeed, transgenic potato plants lacking both St13- AOS genes ( Co AOS1/ 2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in Co AOS1/ 2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and Co AOS1/ 2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii. [ABSTRACT FROM AUTHOR]

Coralallene oxide synthase (cAOS) catalyzes the formation of alleneoxides from fatty acid hydroperoxides. Interestingly, its active sitediffers from that of catalase by only a single residue yet is incapableof catalase activity. That is, it is unable to catalyze the decompositionof hydrogen peroxide to molecular oxygen and water. However, the singleactive-site mutation T66V allows cAOS to exhibit catalase activity.We have performed a series of molecular dynamics (MD) simulationsin order to gain insights into the differences in substrate (8R-hydroperoxyeicosatetraenoic) and H2O2active site binding between wild-type cAOS and the T66V mutant cAOS.It is observed that in wild-type cAOS the active site Thr66 residueconsistently forms a strong hydrogen-bonding interaction with H2O2(catalase substrate) and, importantly, withthe aid of His67 helps to pull H2O2away fromthe heme Fe center. In contrast, in the T66V-cAOS mutant the H2O2is much closer to the heme’s Fe centerand now forms a consistent Fe···O2H2interaction. In addition, the His67···H2O2distance shortens considerably, increasing thelikelihood of a Cpd I intermediate and hence exhibiting catalase activity. [ABSTRACT FROM AUTHOR]

Drought stress is a major limiting factor for crop production. Cuticular wax plays an important role in preventing water loss from drought stress. However, the genetic control of cuticular wax deposition under drought stress conditions has not been characterized. Here, we identified a rice gene Drought-Induced Wax Accumulation 1 (DWA1) encoding a very large protein (2,391 aa in length) containing multiple enzymatic structures, including an oxidoreductase-like domain; a prokaryotic nonribosomal peptide synthetase-like module, including an AMP-binding domain; and an allene oxide synthase-like domain. This previously unreported putative megaenzyme is conserved in vascular plants. A dwa1 KO mutant was highly sensitive to drought stress relative to the WT. DWA1 was preferentially expressed in vascular tissues and epidermal layers and strongly induced by drought stress. The dwa1 mutant was impaired in cuticular wax accumulation under drought stress, which significantly altered the cuticular wax composition of the plant, resulting in increased drought sensitivity. The mutant had reduced levels of very-long-chain fatty acids, and plants overexpressing DWA1 showed elevated levels of very-long-chain fatty acids relative to the WT. The expression of many wax-related genes was significantly suppressed in dwa1 under drought conditions. The AMP-binding domain exhibited in vitro enzymatic activity in activating long-chain fatty acids to form acyl-CoA. Our results suggest that DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice. This finding may have significant implications for improving the drought resistance of crop varieties. [ABSTRACT FROM AUTHOR]

Highlights: [•] Fatty acid TMS-peroxides afford numerous thermal conversion products upon GC–MS. [•] Cyclopentenones were detected first time along with other thermal products. [•] Thermal treatment of fatty acid hydroperoxides in ampoules affords cyclopentenones. [•] Thermal formation of cyclopentenones mimics the allene oxide synthase pathway. [Copyright &y& Elsevier]

Using a recently developed model pathosystem involving Medicago truncatula and Mycosphaerella pinodes, causal agent of Mycosphaerella blight on pea to understand host molecular response to a fungal suppressor, we applied the suppressor to leaves of M. truncatula and identified 151 nonredundant cDNA fragments as newly expressed genes. These included genes encoding lipoxygenase (LOX) and enoyl-CoA hydratase, which are presumably involved in jasmonic acid (JA) synthesis. Potential genes encoding plastidic enzymes, including allene oxide synthase (AOS) and allene oxide cyclase (AOC), and other peroxisomal enzymes involved in β-oxidation were predicted from the Medicago Gene Index EST database and tested for altered expression by semiquantitative RT-PCR. The coordinated expression of genes encoding both plastidic and peroxisomal enzymes showed that the suppressor likely conditions certain cellular process(es) through the JA synthesis in M. truncatula. To explore the role of JA or JA-regulated cellular process(es) in conditioning susceptibility, we used an Apple latent spherical virus (ALSV)-based virus-induced gene silencing (VIGS) technology to silence pea genes including LOX, AOS, AOC and 12- oxo- phytodienoic acid reductase ( OPR). In LOX-, AOS-, AOC- or OPR-silenced pea plants, disease development induced by M. pinodes was remarkably reduced. Similarly, silencing of mRNA for LOX, AOS, AOC or OPR reduced the sensitivity to a phytotoxin, coronatine, which is believed to act through a JA-dependent process. On the basis of these results, it is conceivable that M. pinodes has evolved a strategy to condition susceptibility by manipulating the physiology of host cells, in particular JA-regulated cellular process(es), to promote disease development in pea. [ABSTRACT FROM AUTHOR]

Highlights: [•] The interrelation of CYP74 enzymes was tested by single point mutations. [•] The mutant forms of tobacco and flax divinyl ether synthases (DESs) were prepared. [•] Mutation sites V379F and E292G belong to the ERR-triad and I-helix domains. [•] Both mutations converted DESs into allene oxide synthases (AOSs). [•] DES to AOS conversions caused by single point mutations are described for the first time. [ABSTRACT FROM AUTHOR]

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9Shydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at-15 °C. One matched previously described allene oxides in its UV spectrum (λmax 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec- 10,12Z-dienoate). The second was a novel stereoisomer (UV λmax 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon asZin the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides. [ABSTRACT FROM AUTHOR]

Inthis present work the mechanism by which cAOS catalyzes the formationof allene oxide from its hydroperoxy substrate was computationallyinvestigated by using a DFT-chemical cluster approach. In particular,the effects of dispersion interactions and DFT functional choice (M06,B3LYP, B3LYP*, and BP86), as well as the roles of multistate reactivityand the tyrosyl proximal ligand, were examined. It is observed thatthe computed relative free energies of stationary points along theoverall pathway are sensitive to the choice of DFT functional, whilethe mechanism obtained is generally not. Large reductions in relativefree energies for stationary points along the pathway (compared tothe initial reactant complex) of on average 46.3 and 97.3 kJ mol–1for the doublet and quartet states, respectively,are observed upon going from the M06 to BP86 functional. From resultsobtained by using the B3LYP* method, well-tested previously on heme-containingsystems, the mechanism of cAOS appears to occur with considerablyhigher Gibbs free energies than that for the analogous pathway inpAOS, possibly due to the presence of a ligating tyrosyl residue incAOS. Furthermore, at the IEFPCM-B3LYP*/6-311+G(2df,p)//B3LYP/BS1level of theory the inclusion of dispersion effects leads to the suggestionthat the overall mechanism of cAOS could occur withoutthe need for spin inversion. [ABSTRACT FROM AUTHOR]

Abstract: Jasmonic acid (JA) plays a regulatory role in plant development such as tuber and corm formation and their responses to environmental stresses. The action of JA in regulating plant growth and stress responses often requires the elevation of endogenous levels by de novo synthesis. Allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase 3 (OPR3) are the key enzymes involved in jasmonic acid biosynthesis in plants. Here, we isolated the AOS, AOC and OPR3 genes (GhAOS, GhAOC and GhOPR3) in full length from in vitro developing corms of Gladiolus hybridus. The subcellular localization analysis indicated that both GhAOS and GhAOC were chloroplast localization proteins whereas GhOPR3 was localized within the peroxisome. Real-time quantitative PCR showed that GhAOS, GhAOC and GhOPR3 genes were expressed constitutively in all organs with different levels having a relatively higher level in corms and cormels. Additionally, we observed that MJ treatments contributed to increasing the amount of three genes, mRNA level and endogenous MJ content thus promoting corm formation and enlargement. These results revealed that GhAOS, GhAOC and GhOPR3 were genes of significant importance and their expression contributed to JA biosynthesis thus playing an important role in corm formation and enlargement. [Copyright &y& Elsevier]

Aspergilli oxidize C18 unsaturated fatty acids by dioxygenasecytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy- 10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10- epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn964 may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn878 in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln881 markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln881 with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism. [ABSTRACT FROM AUTHOR]

Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coil-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COIl -dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular "stand-by mode" by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COl1 -dependent leaf growth inhibition. [ABSTRACT FROM AUTHOR]

Insect herbivores have developed a myriad of strategies to manipulate the defense responses of their host plants. Here we provide evidence that chewing insects differentially alter the oxylipin profiles produced by the two main and competing branches of the plant defensive response pathway, the allene oxide synthase ( AOS) and hydroperoxide lyase ( HPL) branches, which are responsible for wound-inducible production of jasmonates ( JAs), and green leafy volatiles ( GLVs) respectively. Specifically, we used three Arabidopsis genotypes that were damaged by mechanical wounding or by insects of various feeding guilds (piercing aphids, generalist chewing caterpillars and specialist chewing caterpillars). We established that emission of GLVs is stimulated by wounding incurred mechanically or by aphids, but release of these volatiles is constitutively impaired by both generalist and specialist chewing insects. Simultaneously, however, these chewing herbivores stimulated JA production, demonstrating targeted insect suppression of the HPL branch of the oxylipin pathway. Use of lines engineered to express HPL constitutively, in conjunction with quantitative RT- PCR-based expression analyses, established a combination of transcriptional and post-transcriptional reprogramming of the HPL pathway genes as the mechanistic basis of insect-mediated suppression of the corresponding metabolites. Feeding studies suggested a potential evolutionary advantage of suppressing GLV production, as caterpillars preferably consumed leaf tissue from plants that had not been primed by these volatile cues. [ABSTRACT FROM AUTHOR]

Abstract: The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand. [Copyright &y& Elsevier]

Abstract: It has previously been shown that jasmonic acid affects the ethylene signaling pathway. EIN2 is a central component of ethylene signaling that is downstream of the receptors. EIN2 has previously been shown to be required for ethylene responses. We found that reducing jasmonic acid levels, either mutationally or chemically, caused ein2 ethylene-insensitive mutants to become ethylene responsive. This effect was not seen with the ethylene-insensitive etr1-1 mutants that affect receptor function. Based upon these results, we propose a model where jasmonic acid is inhibiting ethylene signal transduction down-stream of the ethylene receptors. This may involve an EIN2-independent pathway. [Copyright &y& Elsevier]

Abstract: Peach (Prunus persica) was chosen as a model to further clarify the physiological role of jasmonates (JAs) during fruit ripening. To this aim, the effect of methyl jasmonate (MJ, 0.88mM), applied at a late stage (S3) of fruit development under field conditions (in planta), on the time-course of fruit ripening over a 14-day period was evaluated. As revealed by a non-destructive device called a DA-meter, exogenously applied MJ impaired the progression of ripening leading to less ripe fruit at harvest. To better understand the molecular basis of MJ interference with ripening, the time-course changes in the expression of ethylene-, cell wall-, and auxin-related genes as well as other genes (LOX, AOS and bZIP) was evaluated in the fruit mesocarp. Real-time PCR analyses revealed that transcript levels of ethylene-related genes were strongly affected. In a first phase (days 2 and/or 7) of the MJ response, mRNAs of the ethylene biosynthetic genes ACO1, ACS1 and the receptor gene ETR2 were strongly but transiently down-regulated, and then returned to or above control levels in a second phase (days 11 and/or 14). Auxin biosynthetic, conjugating, transport and perception gene transcripts were also affected. While biosynthetic genes (TRPB and IGPS) were up-regulated, auxin-conjugating (GH3), perception (TIR1) and transport (PIN1) genes were transiently but strongly down-regulated in a first phase, but returned to control levels subsequently. Transcript levels of two JA-related genes (LOX, AOS) and a developmentally regulated transcription factor (bZIP) were also affected, suggesting a shift ahead of the ripening process. Thus, in peach fruit, the transient slowing down of ripening by exogenous MJ was associated with an interference not only with ethylene but also with auxin-related genes. [Copyright &y& Elsevier]

Abstract: We examined the relationship between temperature (15–35°C) and flower induction as it is influenced by linolenic acid (LA) cascade products, lipoxygenase (LOX; EC 1.13.11.12), allene oxide synthase (AOS; EC 4.2.1.92), and allene oxide cyclase (AOC; EC 5.3.99.6) generated in morning glory (Pharbitis nil Choisy). The maximum amount of LOX protein was detected when plants were grown at 30°C, whereas endogenous AOS and AOC proteins were markedly accumulated at 15°C. Although both test levels of 9(S)- and 13(S)-hydroperoxy linolenic acid (HPOT) showed similar temperature dependencies, reflecting the profile of LOX, the relative amount of 13(S)-HPOT was much higher than that of 9(S)-HPOT, regardless of temperature regime. This implied a faster reaction pathway to 9,10-α-ketol octadecadienoic acid (KODA) in the LA cascade. In the 13(S)-HPOT pathway, the highest level of endogenous jasmonic acid (JA) was observed at 15°C. Our results suggest that at a high temperature (30°C), 9(S)-HPOT may be readily metabolized into KODA to promote flower bud formation. By contrast, at a low temperature, high levels of AOS and AOC result in an accumulation of JA that inhibits this developmental process. Accordingly, depending on the growing temperature, flower bud formation in P. nil is possibly regulated by the interactions among LOX metabolites, with KODA serving as a promoter and JA as an inhibitor. [Copyright &y& Elsevier]

Rhomboids are intra-membrane serine proteases whose sequences are found in nearly all organisms. They are involved in a variety of biological functions in both eukaryotes and prokaryotes. Localization assays revealed that two Arabidopsis thaliana rhomboid-like proteases (AtRBL), AtRBL8 and AtRBL9, are targeted to the chloroplast. Using transgenic plants expressing epitope-tagged AtRBL9, we localized AtRBL9 to the chloroplast inner envelope membrane, with both its N- and C-termini facing the stroma. Mass spectrometry analyses confirmed this localization, and suggested that this is also the case for AtRBL8. Both are proteins of very low abundance. The results of size-exclusion chromatography implied that AtRBL9 forms homo-oligomers. In search of a putative function, a comparative proteomic analysis was performed on wild-type and double-knockout plants, lacking both AtRBL8 and AtRBL9, using the iTRAQ method. Of 180 envelope proteins, the level of only a few was either increased or decreased in the mutant line. One of the latter, allene oxide synthase, is involved in jasmonic acid biosynthesis. This observation provides an explanation for the recently reported aberration in flower morphology that is associated with the loss of AtRBL8. [ABSTRACT FROM AUTHOR]

The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is one of the most destructive pests of corn in North America and is currently invading Europe. The two major invasive outbreaks of rootworm in Europe have occurred, in North-West Italy and in Central and South-Eastern Europe. These two outbreaks originated from independent introductions from North America. Secondary contact probably occurred in North Italy between these two outbreaks, in 2008. We used 13 microsatellite markers to conduct a population genetics study, to demonstrate that this geographic contact resulted in a zone of admixture in the Italian region of Veneto. We show that i) genetic variation is greater in the contact zone than in the parental outbreaks; ii) several signs of admixture were detected in some Venetian samples, in a Bayesian analysis of the population structure and in an approximate Bayesian computation analysis of historical scenarios and, finally, iii) allelic frequency clines were observed at microsatellite loci. The contact between the invasive outbreaks in North-West Italy and Central and South-Eastern Europe resulted in a zone of admixture, with particular characteristics. The evolutionary implications of the existence of a zone of admixture in Northern Italy and their possible impact on the invasion success of the western corn rootworm are discussed. [ABSTRACT FROM AUTHOR]

Aims Oxidative stress is an early event in the cascade leading in neuronal damage after hypoxic-ischemic ( HI) brain injury. In the present study, we examined the dose response and window of opportunity for neuroprotection after HI injury with Allene Oxide Synthase ( AOS), an anti-oxidative enzyme of the member of cytochrome P450 family. Methods Adult male rats received intra-cerebro-ventricular infusions of either saline (vehicle) or AOS (1 μg or 10 μg or 100 μg per rat, intracerebroventricular n = 16 all groups) either 45 min or 3 h after unilateral HI brain injury. Brains were collected 5 days later. The extent of brain damage, neuronal survival, apoptosis, and glial reactions were assessed in the striatum, hippocampus, and cortex. Results Allene Oxide Synthase was associated with reduced neuronal damage scores when given 45 min, but not 3 h, after HI injury ( P < 0.0001) in all brain regions. AOS treatment (10 μg) improved neuronal survival in the striatum, cortex, and hippocampus ( P < 0.05, P < 0.001) and reduced the microglia reaction ( P < 0.05) and numbers of caspase-3-positive cells in the hippocampus ( P < 0.01). Conclusions Early blockade of oxidative stress after HI injury reduces inflammatory response, neuronal necrosis, and apoptosis. The neuroprotective effects of AOS were time of administration-dependent suggesting a relatively restricted window of opportunity for acute brain injury. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we demonstrate that NH4 + nutrition in citrange Carrizo plants acts as an inducer of resistance against salinity conditions. We investigated its mode of action and provide evidence that NH4 + confers resistance by priming abscisic acid and polyamines, and enhances H2O2 and proline basal content. Moreover, we observed reduced Cl− uptake as well as enhanced PHGPx expression after salt stress. Control and N-NH4 + plants showed optimal growth. However, N-NH4 + plants displayed greater dry weight and total lateral roots than control plants, but these differences were not observed for primary root length. Our results revealed that N-NH4 + treatment induces a similar phenotypical response to the recent stress-induced morphogenetic response (SIMRs). The hypothesis is that N-NH4 + treatment triggers mild chronic stress in citrange Carrizo plants, which might explain the SIMR observed. Moreover, we observed modulators of stress signaling, such as H2O2 in N-NH4 + plants, which could acts as an intermediary between stress and the development of the SIMR phenotype. This observation suggests that NH4 + treatments induce a mild stress condition that primes the citrange Carrizo defense response by stress imprinting and confers protection against subsequent salt stress. [Copyright &y& Elsevier]

The ability of hemoproteins to catalyze epoxidation or hydroxylation reactions is usually associated with a cysteine as the proximal ligand to the heme, as in cytochrome P450 or nitric oxide synthase. Catalase-related allene oxide synthase (cAOS) from the coral Plexaura homomalla, like catalase itself, has tyrosine as the proximal heme ligand. Its natural reaction is to convert 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE) to an allene epoxide, a reaction activated by the ferric heme, forming product via the FeIV-OH intermediate, Compound II. Here we oxidized cAOS to Compound I (FeV=O) using the oxygen donor iodosylbenzene and investigated the catalytic competence of the enzyme. 8R-hydroxyeicosatetraenoic acid (8R-HETE), the hydroxy analog of the natural substrate, normally unreactive with cAOS, was thereby epoxidized stereospecifically on the 9,10 double bond to form 8R-hydroxy-9R,10R-trans-epoxy-eicosa- 5Z,11Z,14Z-trienoic acid as the predominant product; the turnover was 1/s using 100 μMiodosylbenzene. The enantiomer, 8S-HETE, was epoxidized stereospecifically, although with less regiospecificity, and was hydroxylated on the 13- and 16-carbons. Arachidonic acid was converted to two major products, 8R-HETE and 8R,9S-eicosatrienoic acid (8R,9S-EET), plus other chiral monoepoxides and bis-allylic 10S-HETE. Linoleic acid was epoxidized, whereas stearic acid was not metabolized. We conclude that when cAOS is charged with an oxygen donor, it can act as a stereospecific monooxygenase. Our results indicate that in the tyrosine-liganded cAOS, a catalase-related hemoprotein in which a polyunsaturated fatty acid can enter the active site, the enzyme has the potential to mimic the activities of typical P450 epoxygenases and some capabilities of P450 hydroxylases. [ABSTRACT FROM AUTHOR]

When plants are under insect herbivore attack defensive measures are activated not only locally, but also in distant and systemic tissues. While insect elicitors (IE) abundant in the oral secretions of the attacking herbivore are essential in the regulation of induced defenses, little is known about their effects on systemic defense signaling in maize (Zea mays). The goal of this study was therefore to identify genetic markers that can be used to further characterize local and systemic signaling events induced by IE or mechanical wounding (MW). We selected genes for this study based on their putative involvement in signaling (allene oxide synthase), regulation of gene expression (transcription factor MYC7), and in direct defenses (ribosome inactivating protein) and analyzed their expression in different sections of the treated leaf as well as in systemic parts of the same plant. We found the most significant transcript accumulation of the selected genes after treatment with insect elicitors in those parts with increased JA levels. Additionally, treatment with IE did also induce the accumulation of MYC7 transcripts in basal parts of the treated leaf and systemically. MW, in contrast, did induce RIP and AOS only locally, but not MYC7. This local suppression of MYC7 was further studied by adding glutathione (GSH) as an electron donor to MW plants to quench putative α, β-unsaturated carbonyls, which build up to significant levels around the damage site. Indeed, GSH-treated MW plants accumulated MYC7 at the damage site and also produced more volatiles, suggesting a putative redox-regulatory element being involved in the suppression of MYC7. The results presented herein provide evidence for the specific induction of distant signaling events triggered by IE, most likely through electric signaling. Additionally, a putative role for MW-induced α, β-unsaturated carbonyls in the transcriptional regulation of defense genes was discovered. [ABSTRACT FROM AUTHOR]

A series of new triazole derivatives was synthesized and their inhibitory activity against allene oxide synthase (AOS, CYP74A), a key enzyme in jasmonic acid biosynthesis, was evaluated. Structure-activity relationship studies revealed that methyl 8-[1-(naphthalen-2-yl)-2-(1,2,4-triazol-1-yl) ethoxy]octanate (4i) and methyl 8-[1-(2,4-dichlorophenyl)-2-(1,2,4-triazol- 1-yl)ethoxy]octanate (4g) exhibit potent inhibitory activity to allene oxide synthase, with IC50 values of 0.75±0.30 and 0.84±0.60 µM, respectively. [ABSTRACT FROM AUTHOR]

Allene oxide synthase (AOS) is the first committed step in the biosynthetic pathway of Jasmonate. In this study, a full-length cDNA of AOS gene (named as AaAOS) was cloned from Artemisia annua. The gene was 1891 bp in size containing an open reading frame (1581 bp) encoding 526 amino acids. Comparative and bioinformatic analysis revealed that the deduced protein of AaAOS was highly homologous to AOSs from other plant species. Phylogenetic analysis indicated that the protein of AaAOS belonged to the dicotyledonous group, which was consistent with the category of A. annua. Southern blot analysis revealed that it was a low-copy gene. Quantitative Real-time PCR (qRT-PCR) analysis showed that AaAOS mRNA accumulated most abundantly in leaves and flowers. The qRT-PCR analysis revealed that MeJA, ABA and ethylene treatments significantly enhanced AaAOS transcript expression. [ABSTRACT FROM AUTHOR]

Background: The moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. Results: Our results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis. Conclusions: Our study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes. [ABSTRACT FROM AUTHOR]

Plant oxylipins are involved in defense responses against pathogens or herbivores. Each oxylipin compound exerts its distinctive physiological function. The ability of many legumes to fix nitrogen with rhizobia gives them special importance in natural environments and agriculture. It has been postulated that oxylipins are somehow related to symbiosis; however, this is still a controversial issue. In this study, we isolated five genes at the branching point of the oxylipin pathway in Lotus japonicus, and their biochemical functions were identified as allene oxide synthases (AOSs), 13-hydroperoxide lyase (13HPL), and 9/13-HPLs. When the leaves were mechanically wounded, AOS and 9/13HPL were upregulated in leaves and roots, respectively, from which their implications in wound response were suggested. When the plants were inoculated with rhizobia, no big change in the expression levels of genes was found. When high N was supplied to the nodulated plants, the number of nodules decreased, and simultaneously, AOS in the leaves was downregulated. Significance of AOS in response to the N status in the plants was suggested. [ABSTRACT FROM PUBLISHER]

Lipoxygenase (LOX) and allene oxide synthase (AOS) are key enzymes in the jasmonic acid (JA) biosynthesis pathway. In this work, the role of LOX and AOS in defense response induced by wounding was investigated using pea ( Pisum sativum L., cv. Ning Xia) seedlings as the material. The results showed that wound-induced 'JA burst' was accompanied by the activation of LOX and AOS and the accumulation of their mRNAs; applied JA also stimulated the accumulation of LOX and AOS. Further experiments conducted with inhibitors demonstrated that the wound-induced JA was regulated by LOX and AOS at both transcriptional and enzymic levels. Their activation was necessary in wound-mediated defense response and could enhance the tolerance of pea seedlings to wounding. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: To evaluate the importance of the oxylipin pathway in plant defense against viruses we studied the induction of this pathway in a compatible and an incompatible virus–pepper interaction. Obuda pepper virus (ObPV) inoculation led to the appearance of hypersensitive necrotic lesions (incompatible interaction), to a marked induction of lipoxygenase (LOX) enzyme activity, and to elevated transcription of three LOX genes in leaves of the pepper cultivar TL 1791 harbouring the L 3 resistance gene. In the same pepper cultivar Pepper mild mottle virus (PMMoV) inoculation caused very mild chlorotic symptoms (compatible interaction), and exerted only negligible effects on LOX activity and gene expression. The transcription of an allene oxide synthase (CaAOS) and a hydroperoxide lyase (CaHPL) gene was not modified significantly by any virus inoculation. The expression of a novel divinyl ether synthase (CaDES) gene was massively induced in ObPV-inoculated leaves, but only very slightly by PMMoV inoculation. Our results showed that LOX and DES genes may be specifically involved in resistance responses against virus infections. [Copyright &y& Elsevier]

The article presents the study that examines allene oxide synthase (AOS), an enzyme of jasmonic acid (JA) biosynthesis, found in Physcomitrella patens. Presence of Jasmonic acid (JA) was observed in P. patens which are much higher than in flowering plants. The lipoxygenase activity and AOS activity indicates presence of octadecanoid pathway in P. patens. Moreover, it states that JA can act a signal molecule in bryophytes.

Abstract: Jasmonates are a growing class of lipid-derived signaling molecules with diverse functions ranging from the initiation of biotic and abiotic stress responses to the regulation of plant growth and development. Jasmonate biosynthesis originates from polyunsaturated fatty acids in chloroplast membranes. In a first lipoxygenase-catalyzed reaction molecular oxygen is introduced to yield their 13-hydroperoxy derivatives. These fatty acid hydroperoxides are converted by allene oxide synthase and allene oxide cyclase to 12-oxophytodienoic acid (OPDA) and dinor-OPDA, i.e. the first cyclic intermediates of the pathway. In the subsequent step, the characteristic cyclopentanone ring structure of jasmonates is established by OPDA reductase. Until recently, jasmonic acid has been viewed as the end product of the pathway and as the bioactive hormone. It becomes increasingly clear, however, that biological activity extends to and may even differ between the various jasmonic acid metabolites and conjugates as well as its biosynthetic precursors. It has also become clear that oxygenated fatty acids give rise to a vast variety of bioactive compounds including but not limited to jasmonates. Recent insights into the structure, function, and regulation of the enzymes involved in jasmonate biosynthesis help to explain how this variety is generated while specificity is maintained. [Copyright &y& Elsevier]

Abstract: The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a “different look” at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As enzyme reactions and P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture. [Copyright &y& Elsevier]

Nonclassical P450s of the CYP74 family catalyse the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. The model organism, spikemoss Selaginella moellendorffii Hieron, possesses at least ten CYP74 genes of novel J, K, L, and M subfamilies. The cloning of three CYP74L genes and catalytic properties of recombinant proteins are described in the present work. The CYP74L1 possessed mainly hydroperoxide lyase (HPL) activity towards the 13(S)-hydroperoxide of α−linolenic acids (13-HPOT) and nearly equal HPL and allene oxide synthase (AOS) activities towards the 13(S)-hydroperoxide of linoleic acids (13-HPOD). The 9-hydroperoxides were poor substrates for CYP74L1 and led to the production of mainly the α−ketols (AOS products) and minorities of HPL and epoxyalcohol synthase (EAS) products. The CYP74L2 possessed the AOS activity towards all tested hydroperoxides. CYP74L3 possessed low HPL/EAS activity. Besides, the aerial parts of S. moellendorffii plants possessed complex oxylipins patterns including divinyl ethers, epoxyalcohols, and 12-oxo-phytodienoic acid. Characterization of the CYP74L enzymes and oxylipin pattern updates the knowledge on the complex oxylipin biosynthetic machinery in the surviving oldest taxa of vascular plants. [Display omitted] • The coding sequences of Selaginella moellendorffii CYP74L genes have been cloned. • The recombinant CYP74L1 possesses mainly hydroperoxide lyase activity. • The recombinant CYP74L2 exhibits allene oxide synthase activity. • CYP74Ls append the complex picture of oxylipin biosynthesis in oldest land plants. [ABSTRACT FROM AUTHOR]

Abstract: Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the “I-helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis. [Copyright &y& Elsevier]

Abstract: A catalase-related allene oxide synthase (cAOS) and true catalases that metabolize hydrogen peroxide have similar structure around the heme. One of the distal heme residues considered to help control catalysis is a highly conserved asparagine. Here we addressed the role of this residue in metabolism of the natural substrate 8R-hydroperoxyeicosatetraenoic acid by cAOS and in H2O2 breakdown by catalase. In cAOS, the mutations N137A, N137Q, N137S, N137D, and N137H drastically reduced the rate of reaction (to 0.8–4% of wild-type), yet the mutants all formed the allene oxide as product. This is remarkable because there are many potential heme-catalyzed transformations of fatty acid hydroperoxides and special enzymatic control must be required. In human catalase, the N148A, N148S, or N148D mutations only reduced rates to ∼20% of wild-type. The distal heme Asn is not essential in either catalase or cAOS. Its conservation throughout evolution may relate to a role in optimizing catalysis. [Copyright &y& Elsevier]