RESUMEN
The product specificity and mechanistic peculiarities of two allene oxide synthases, tomato LeAOS3 (CYP74C3) and maize ZmAOS (CYP74A19), were studied. Enzymes were vortexed with linoleic acid 9-hydroperoxide in a hexane-water biphasic system (20-60 s, 0 °C). Synthesized allene oxide (9,10-epoxy-10,12-octadecadienoic acid; 9,10-EOD) was trapped with ethanol. Incubations with ZmAOS produced predominantly 9,10-EOD, which was converted into an ethanolysis product, (12Z)-9-ethoxy-10-oxo-12-octadecenoic acid. LeAOS3 produced the same trapping product and 9(R)-α-ketol at nearly equimolar yields. Thus, both α-ketol and 9,10-EOD appeared to be kinetically controlled LeAOS3 products. NMR data for 9,10-EOD (Me) preparations revealed that ZmAOS specifically synthesized 10(E)-9,10-EOD, whereas LeAOS3 produced a roughly 4:1 mixture of 10(E) and 10(Z) isomers. The cyclopentenone cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) and the Favorskii-type product yields were appreciable with LeAOS3, but dramatically lower with ZmAOS. The 9,10-EOD (free acid) kept in hexane transformed into macrolactones but did not cyclize. LeAOS3 catalysis is supposed to produce a higher proportion of oxyallyl diradical (a valence tautomer of allene oxide), which is a direct precursor of both cyclopentenone and cyclopropanone. This may explain the substantial yields of cis-10-oxo-PEA and the Favorskii-type product (via cyclopropanone) with LeAOS3. Furthermore, 10(Z)-9,10-EOD may be produced via the reverse formation of allene oxide from oxyallyl diradical.
Asunto(s)
Óxidos , Solanum lycopersicum , Zea mays , HexanosRESUMEN
The genome of the cabbage clubroot pathogen Plasmodiophora brassicae Woronin 1877 (Cercozoa, Rhizaria, SAR), possesses two expressed genes encoding the P450s that are phylogenetically related to the enzymes of oxylipin biosynthesis of the CYP74 clan. The cDNA of one of these genes (CYP50918A1) has been expressed in E. coli. The preferred substrate for the recombinant protein, the 13-hydroperoxide of α-linolenic acid (13-HPOT), was converted to the novel heterobicyclic oxylipins, plasmodiophorols A and B (1 and 2) at the ratio ca. 12:1. Compounds 1 and 2 were identified as the substituted 6-oxabicyclo[3.1.0]hexane and 2-oxabicyclo[2.2.1]heptane (respectively) using the MS and NMR spectroscopy, as well as the chemical treatments. The 18O labelling experiments revealed the incorporation of a single 18O atom from [18O2]13-HPOT into the epoxide and ether functions of products 1 and 2 (respectively), but not into their OH groups. In contrast, the 18O from [18O2]water was incorporated only into the hydroxyl functions. One more minor polar product, plasmodiophorol C (3), identified as the cyclopentanediol, was formed through the hydrolysis of compounds 1 and 2. Plasmodiophorols A-C are the congeners of egregiachlorides, hybridalactone, ecklonialactones and related bicyclic oxylipins detected before in some brown and red algae. The mechanism of 13-HPOT conversions to plasmodiophorols A and B involving the epoxyallylic cation intermediate is proposed. The hydroperoxide bicyclase CYP50918A1 is the first enzyme controlling this kind of fatty acid hydroperoxide conversion.
Asunto(s)
Peróxidos Lipídicos/genética , Oxilipinas/metabolismo , Plasmodiophorida/genética , Prostaglandina-Endoperóxido Sintasas/genética , Brassica/genética , Brassica/microbiología , Peróxido de Hidrógeno/metabolismo , Peróxidos Lipídicos/metabolismo , Plasmodiophorida/enzimología , Plasmodiophorida/patogenicidad , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/aislamiento & purificaciónRESUMEN
A series of conjugates of diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) and N-acetyl-D-glucosamine was synthesised and their cytotoxicity against several human cancer cell lines (M-Hela, MCF-7, Hep G2, Panc-1, PC-3), as well as normal human cell lines (WI-38, Chang liver) was assayed. Most of the conjugates were found to be cytotoxic against the mentioned cancer cell lines in the range of IC50 values 13-89 µM. Two lead compounds 14a and 14b showed selective cytotoxicity against M-Hela (IC50 13 and 14 µM) that was two times as high as the cytotoxicity of the anti-cancer drug Tamoxifen in control (IC50 28 µM). It was found that cytotoxic activity of the lead compounds against M-Hela cells is due to induction of apoptosis.
Asunto(s)
Acetilglucosamina/síntesis química , Acetilglucosamina/farmacología , Diterpenos de Tipo Kaurano/síntesis química , Diterpenos de Tipo Kaurano/farmacología , Diterpenos/síntesis química , Diterpenos/farmacología , Acetilglucosamina/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Diterpenos/química , Diterpenos de Tipo Kaurano/química , Ensayos de Selección de Medicamentos Antitumorales , Hemólisis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Relación Estructura-ActividadRESUMEN
The model moss Physcomitrella patens and liverwort Marchantia polymorpha possess all enzymatic machinery responsible for the initial stages of jasmonate pathway, including the active 13(S)-lipoxygenase, allene oxide synthase (AOS) and allene oxide cyclase (AOC). At the same time, the jasmonic acid is missing from both P. patens and M. polymorpha. Our GC-MS profiling of oxylipins of P. patens gametophores and M. polymorpha tissues revealed some distinctive peculiarities. The 15(Z)-cis-12-oxo-10,15-phytodienoic acid (12-OPDA) was the major oxylipin in M. polymorpha. In contrast, the 12-OPDA was only a minor constituent in P. patens, while another cyclopentenone 1 was the predominant oxylipin. Product 1 was identified by its MS, 1H-NMR, 1H-1H-COSY, HSQC and HMBC data as 15(Z)-12-oxo-9(13),15-phytodienoic acid, i.e., the iso-12-OPDA. The corresponding C16 homologue, 2,3-dinor-iso-12-OPDA (2), have also been detected as a minor component in P. patens and a prominent product in M. polymorpha. Besides, the 2,3-dinor-cis-12-OPDA (3) was also present in M. polymorpha. Apparently, the malfunction of cyclopentenone reduction by the 12-OPDA reductase in P. patens and (to a lesser extent) in M. polymorpha leads to the isomerization of 12-OPDA and formation of specific cyclopentenones 1 and 2, which are uncommon in flowering plants.
Asunto(s)
Bryopsida , Marchantia , Ciclopentanos , Ácidos Grasos Insaturados , Lipooxigenasa , Marchantia/genética , OxilipinasRESUMEN
Screening of linolipins, i.e. galactolipids containing esterified residues of divinyl ether oxylipins, in the leaves of several higher plants revealed the presence of these complex oxylipins in the meadow buttercup leaves. The rapid accumulation of linolipins occurred in the injured leaves of meadow buttercup, while intact leaves possessed no linolipins. These oxylipins were isolated from the injured leaves, separated and purified by HPLC. The structural analyses of linolipins by UV, mass-spectroscopy and NMR spectroscopy resulted in the identification of eight molecular species. Three of them were identical to linolipins B-D found earlier in the leaves of flax (Linum usitatissimum L.). Other molecular species were identified as 1-O-(ω5Z)-etherolenoyl-2-O-dinor-(ω5Z)-etherolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, 1-O-(ω5Z)-etherolenoyl-2-O-(7Z,10Z,13Z)-hexadecatrienoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, 1-O-(ω5Z)-etherolenoyl-2-O-(7Z,10Z)-hexadecadienoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, 1-O-(ω5Z)-etherolenoyl-2-O-α-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, and 1-O-(ω5Z)-etherolenoyl-2-O-palmitoyl-3-O-(α-galactopyranosyl-1-6-ß-D-galactopyranosyl)-sn-glycerol. The trivial names "linolipins E, F, G, H and I," respectively, have been ascribed to these novel complex oxylipins.
Asunto(s)
Oxilipinas/química , Oxilipinas/aislamiento & purificación , Hojas de la Planta/química , Ranunculus/químicaRESUMEN
Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9(S)hydroperoxide (9(S)HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60â¯s at 0⯰C, pHâ¯4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC-MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9hydroxy9[(1'E,3'Z)nonadienyloxy]nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [13C18]9(S)HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name "hydroperoxide lyase", which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with "hemiacetal synthase" (HAS).
Asunto(s)
Aldehído-Liasas/metabolismo , Cucurbitaceae/enzimología , Peróxidos Lipídicos/química , Cromatografía de Gases y Espectrometría de Masas , Ácidos Linoleicos/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Proteínas de Plantas/metabolismo , Especificidad por SustratoRESUMEN
Young roots of wheat, barley, and sorghum, as well as methyl jasmonate pretreated rice seedlings, undergo an unprecedented allene oxide synthase pathway targeted to previously unknown oxylipins 1-3. These Favorskii-type products, (4Z)-2-pentyl-4-tridecene-1,13-dioic acid (1), (2'Z)-2-(2'-octenyl)-decane-1,10-dioic acid (2), and (2'Z,5'Z)-2-(2',5'-octadienyl)-decane-1,10-dioic acid (3), have a carboxy function at the side chain, as revealed by their MS and NMR spectral data. Compounds 1-3 were the major oxylipins detected, along with the related α-ketols. Products 1-3 were biosynthesized from (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid, (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD), and (9S,10E,12Z,15Z)-9-hydroperoxy-10,12,15-octadecatrienoic acid, respectively, via the corresponding allene oxides and cyclopropanones. The data indicate that conversion of the allene oxide into the cyclopropanone is controlled by soluble cyclase. The short-lived cyclopropanones are hydrolyzed to products 1-3. The collective name "graminoxins" has been ascribed to oxylipins 1-3.
RESUMEN
Plants have developed a complex defense response system against pests and pathogens. Defensins, produced by plants as part of their innate immune response, form the family of small, basic, cysteine-rich proteins with activity primarily directed against fungal pathogens. In addition, plant defensins can show antibacterial activity and protease and insect amylase inhibitory activities. However, in gymnosperms, only antifungal activity of defensins has been described thus far. Here, we report antibacterial and insect α-amylase inhibition activities for defensin PsDef1 from P. sylvestris, the first defensin from gymnosperms with a broad range of biological activities described. We also report the solution NMR structure of PsDef1 and its dynamics properties assessed by a combination of experimental NMR and computational techniques. Collectively, our data provide an insight into structure, dynamics, and functional properties of PsDef1 that could be common between defensins from this taxonomic group.
Asunto(s)
Defensinas/química , Defensinas/farmacología , Pinus sylvestris/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Alineación de Secuencia , alfa-Amilasas/metabolismoRESUMEN
Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [18O2]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Epoxi/metabolismo , Oxilipinas/metabolismo , Phaeophyceae/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Peróxido de Hidrógeno/metabolismo , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/metabolismo , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Ácido alfa-LinolénicoRESUMEN
Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography-mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the (1)H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.
Asunto(s)
Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Selaginellaceae/enzimología , Compuestos de Vinilo/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/genética , Cromatografía de Gases y Espectrometría de Masas , Cinética , Ácidos Linoleicos/metabolismo , Ácidos Linolénicos/metabolismo , Peróxidos Lipídicos/metabolismo , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/genética , Proteínas Recombinantes/metabolismo , Selaginellaceae/clasificación , Selaginellaceae/genética , Especificidad por SustratoRESUMEN
Defensins are part of the innate immune system in plants with activity against a broad range of pathogens, including bacteria, fungi and viruses. Several defensins from conifers, including Scots pine defensin 1 (Pinus sylvestris defensin 1, (PsDef1)) have shown a strong antifungal activity, however structural and physico-chemical properties of the family, needed for establishing the structure-dynamics-function relationships, remain poorly characterized. We use several spectroscopic and computational methods to characterize the structure, dynamics, and oligomeric state of PsDef1. The three-dimensional structure was modeled by comparative modeling using several programs (Geno3D, SWISS-MODEL, I-TASSER, Phyre(2), and FUGUE) and verified by circular dichroism (CD) and infrared (FTIR) spectroscopy. Furthermore, FTIR data indicates that the structure of PsDef1 is highly resistant to high temperatures. NMR diffusion experiments show that defensin exists in solution in the equilibrium between monomers and dimers. Four types of dimers were constructed using the HADDOCK program and compared to the known dimer structures of other plant defensins. Gaussian network model was used to characterize the internal dynamics of PsDef1 in monomer and dimer states. PsDef1 is a typical representative of P. sylvestris defensins and hence the results of this study are applicable to other members of the family.
Asunto(s)
Defensinas/química , Modelos Moleculares , Pinus sylvestris/química , Proteínas de Plantas/química , Conformación Proteica , Secuencia de Aminoácidos , Dicroismo Circular , Datos de Secuencia Molecular , Posición Específica de Matrices de Puntuación , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Espectroscopía de Protones por Resonancia Magnética , Proteínas Recombinantes , Alineación de Secuencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5-Cys31 and Cys7-Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer.
Asunto(s)
beta-Tromboglobulina/química , beta-Tromboglobulina/metabolismo , Dicroismo Circular , Disulfuros , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Estabilidad Proteica , Desplegamiento Proteico , TermodinámicaRESUMEN
Enzymes of the CYP74 family, including the divinyl ether synthase (DES), play important roles in plant cell signalling and defence. The potent DES activities have been detected before in the leaves of the meadow buttercup (Ranunculus acris L.) and few other Ranunculaceae species. The nature of these DESs and their genes remained unrevealed. The PCR with degenerate primers enabled to detect the transcript of unknown P450 gene assigned as CYP74Q1. Besides, two more CYP74Q1 isoforms with minimal sequence variations have been found. The full length recombinant CYP74Q1 protein was expressed in Escherichia coli. The preferred substrates of this enzyme are the 13-hydroperoxides of α-linolenic and linoleic acids, which are converted to the divinyl ether oxylipins (ω5Z)-etherolenic acid, (9Z,11E)-12-[(1'Z,3'Z)-hexadienyloxy]-9,11-dodecadienoic acid, and (ω5Z)-etheroleic acid, (9Z,11E)-12-[(1'Z)-hexenyloxy]-9,11-dodecadienoic acid, respectively, as revealed by the data of mass spectrometry, NMR and UV spectroscopy. Thus, CYP74Q1 protein was identified as the R. acris DES (RaDES), a novel DES type and the opening member of new CYP74Q subfamily.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Filogenia , Hojas de la Planta/química , Proteínas de Plantas/metabolismo , Ranunculus/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Sistema Enzimático del Citocromo P-450/clasificación , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ácidos Linoleicos/metabolismo , Datos de Secuencia Molecular , Oxilipinas/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa , Ranunculus/enzimología , Ranunculus/genética , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Ácido alfa-Linolénico/metabolismoRESUMEN
This manuscript has been withdrawn by the Author.
RESUMEN
Two complex oxylipins (linolipins C and D) were isolated from the leaves of flax plants inoculated with phytopathogenic bacteria Pectobacterium atrosepticum. Their structures were elucidated based on UV, MS and NMR spectroscopic data. Both oxylipins were identified as digalactosyldiacylglycerol (DGDG) molecular species. Linolipin C contains one residue of divinyl ether (ω5Z)-etherolenic acid and one α-linolenate residue at sn-1 and sn-2 positions, respectively. Linolipin D possesses two (ω5Z)-etherolenic acid residues at both sn-1 and sn-2 positions. The rapid formation (2-30min) of linolipins C and D alongside with linolipins A and B occurred in the flax leaves upon their damage by freezing-thawing.
Asunto(s)
Lino/química , Oxilipinas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Lino/metabolismo , Lino/microbiología , Galactolípidos/química , Lipasa/metabolismo , Modelos Moleculares , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oxilipinas/química , Pectobacterium/metabolismo , Hojas de la Planta/química , Hojas de la Planta/microbiología , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Estereoisomerismo , Ácido alfa-Linolénico/análisisRESUMEN
The approach based on analysis of the residual 1H-13C dipolar couplings in molecules partially aligned in a lyotropic liquid crystalline medium was used in the NMR investigation of the reduced glutathione (Glu-Cys-Gly; GSH) structure in a lyotropic medium (cetylpyridinium chloride-n-hexanol). The spatial structure of GSH in solution was established on the basis of the experimental data for observed couplings only.