CYP77A19 and CYP77A20 characterized from Solanum tuberosum oxidize fatty acids in vitro and partially restore the wild phenotype in an Arabidopsis thaliana cutin mutant.

Cutin and suberin represent lipophilic polymers forming plant/environment interfaces in leaves and roots. Despite recent progress in Arabidopsis, there is still a lack on information concerning cutin and suberin synthesis, especially in crops. Based on sequence homology, we isolated two cDNA clones of new cytochrome P450s, CYP77A19 and CYP77A20 from potato tubers (Solanum tuberosum). Both enzymes hydroxylated lauric acid (C12:0) on position ω-1 to ω-5. They oxidized fatty acids with chain length ranging from C12 to C18 and catalysed hydroxylation of 16-hydroxypalmitic acid leading to dihydroxypalmitic (DHP) acids, the major C16 cutin and suberin monomers. CYP77A19 also produced epoxides from linoleic acid (C18:2). Exploration of expression pattern in potato by RT-qPCR revealed the presence of transcripts in all tissues tested with the highest expression in the seed compared with leaves. Water stress enhanced their expression level in roots but not in leaves. Application of methyl jasmonate specifically induced CYP77A19 expression. Expression of either gene in the Arabidopsis null mutant cyp77a6-1 defective in flower cutin restored petal cuticular impermeability. Nanoridges were also observed in CYP77A20-expressing lines. However, only very low levels of the major flower cutin monomer 10,16-dihydroxypalmitate and no C18 epoxy monomers were found in the cutin of the complemented lines.

Specific accumulation of CYP94A1 transcripts after exposure to gaseous benzaldehyde: induction of lauric acid ω-hydroxylase activity in Vicia sativa exposed to atmospheric pollutants.

The effects of air pollutants such as aldehydes, ozone, nitrogen dioxide and benzene on fatty acid ω-hydroxylase activity in Vicia sativa microsomes have been investigated. Four days old etiolated V. sativa seedlings were exposed to different concentrations of selected pollutants for varying exposure times. Growing etiolated V. sativa seedlings in air containing the gaseous benzaldehyde (150 nM) led to an 8-fold enhancement of lauric acid ω-hydroxylase activity in microsomes of treated plants compared to controls grown in pure air (96 ± 10 versus 12 ± 2 pmol/min/mg protein, respectively). The induction increased with increasing gas phase concentrations (10-1300 nM) and the maximum of activity was measured after 48 h of exposure. Northern blot analysis revealed that this induction occurred via transcriptional activation of the gene coding for CYP94A1. The absence of CYP94A2 and CYP94A3 transcription activation together with the missing effect on epoxide hydrolases activities indicate the specificity of CYP94A1 induction by benzaldehyde. Exposure to nitrogen dioxide, ozone and formaldehyde also stimulated lauric acid ω-hydroxylases activity while exposure to benzene did not show any effect.

Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega -hydroxylation in development.

We describe lacerata (lcr) mutants of Arabidopsis, which display various developmental abnormalities, including postgenital organ fusions, and report cloning of the LCR gene by using the maize transposon Enhancer/Suppressor-mutator (En/Spm). The pleiotropic mutant phenotype could be rescued by genetic complementation of lcr mutants with the wild-type LCR gene. The LCR gene encodes a cytochrome P450 monooxygenase, CYP86A8, which catalyzes omega-hydroxylation of fatty acids ranging from C12 to C18:1, as demonstrated by expression of the gene in yeast. Although palmitic and oleic acids were efficient substrates for LCR, 9,10-epoxystearate was not metabolized. Taken together with previous studies, our findings indicate that LCR-dependent omega-hydroxylation of fatty acids could be implicated in the biosynthesis of cutin in the epidermis and in preventing postgenital organ fusions. Strikingly, the same pathway seems to control trichome differentiation, the establishment of apical dominance, and senescence in plants.

A conservative amino acid substitution alters the regiospecificity of CYP94A2, a fatty acid hydroxylase from the plant Vicia sativa.

Fatty acid omega-hydroxylation is involved in the biosynthesis of the plant cuticle, formation of plant defense signaling molecules, and possibly in the rapid catabolism of free fatty acids liberated under stress conditions. CYP94A2 is a cytochrome P450-dependent medium-chain fatty acid hydroxylase that was recently isolated from Vicia sativa. Contrary to CYP94A1 and CYP86A1, two other fatty acid hydroxylases previously characterized in V. sativa and Arabidopsis thaliana, CYP94A2 is not a strict omega-hydroxylase, but exhibits chain-length-dependent regioselectivity of oxidative attack. Sequence alignments of CYP94A2 with CYP94A1 and molecular modeling studies suggested that F494, located in SRS-6 (substrate recognition site) was involved in substrate recognition and positioning. Indeed, a conservative amino acid substitution at that position markedly altered the regiospecificity of CYP94A2. The observed shift from omega toward omega-1 hydroxylation was prominent with lauric acid as substrate and declined with increasing fatty acid chain length.

CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.

Cress and potato soluble epoxide hydrolases: purification, biochemical characterization, and comparison to mammalian enzymes.

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

Adaptive responses of human monocytes infected by bordetella pertussis: the role of adenylate cyclase hemolysin.

The activation/adaptive responses of human monocytes exposed to Bordetella pertussis parental or mutant strains were evaluated and correlated to the expression of two bacterial toxins: adenylate cyclase-hemolysin and pertussis toxin. The marked rise in intracellular cyclic adenosine monophosphate (cAMP) observed in monocytes infected by B. pertussis parental strain, inversely correlated with (1) the production of tumor necrosis factor alpha; (2) the release of superoxide anion; and (3) the expression of the 72-kDa heat shock/stress protein, Hsp70. Experiments performed with mutants deficient in adenylate cyclase-hemolysin or with purified bacterial toxins confirmed the key role of adenylate cyclase-hemolysin in the control of monocytes' response to infection by B. pertussis. This bacterial strategy primarily involves evasion from antimicrobial defenses and, eventually, the sacrifice of the host cell.

Curosurf modulates cAMP accumulation in human monocytes through a membrane-controlled mechanism.

The cellular mechanisms by which pulmonary surfactant exerts its effects, including anti-inflammatory or proinflammatory effects, have remained elusive. To address the issue of whether plasma membrane modifications represent a target for these mechanisms, we designed an experimental protocol involving the determination of changes in cAMP levels under membrane-dependent or -independent stimulatory pathways. The effects of a modified natural porcine surfactant, Curosurf, and the major surfactant protein A were evaluated on resting and stimulated cAMP levels of human monocytes. We found that agents that elevate intracellular cAMP exhibit different susceptibilities toward a preexposure to Curosurf. The rise in cAMP induced by membrane-active agents such as cholera toxin or the diterpene forskolin was significantly inhibited by monocyte preexposure to Curosurf. In contrast, the rise in cAMP induced by the membrane-permeant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or by the Bordetella pertussis toxin adenylate cyclase-hemolysin was unaffected by Curosurf. Surfactant protein A did not affect either cAMP levels or the inhibitory capacity of Curosurf. We suggest that a plasma membrane-associated event affecting the mechanism underlying the effects of cholera toxin or forskolin is involved in the inhibition of cAMP accumulation caused by Curosurf.

omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence.

The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.

Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant-pathogen interactions: enantioselectivity studies.

The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids. These compounds are also known messengers in plant-pathogen interactions. We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaün, Bosch, Lesot, Mioskowski and Durst (1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives. The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450. Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9 and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol.

Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material. Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different.

Modified natural porcine surfactant modulates tobacco smoke-induced stress response in human monocytes.

Tobacco smoke (TS) is a potent source of oxidants and oxidative stress is an important mechanism by which TS exerts its toxicity in the lung. We have shown that TS induces heat shock (HS)/stress protein (HSP) synthesis in human monocytes. Pulmonary surfactant (PS) whose major physiological function is to confer mechanical stability to alveoli, also modulates oxidative metabolism and other pro-inflammatory functions of monocytes-macrophages. In order to determine whether PS alters the stress response induced by TS, we incubated human peripheral blood monocytes overnight with modified natural porcine surfactant (Curosurf) (1 mg/ml) before exposure to TS. Curosurf decreased TS-induced, but not HS-induced, expression of the major cytosolic, inducible 72 kD HSP (Hsp70). Furthermore, TS-generated superoxide anions production was significantly decreased by Curosurf in an acellular system, suggesting a direct scavenging effect of PS. We also examined the effects of TS and PS on monocytes ultrastructure. Monocytes incubated with Curosurf presented smoother cell membranes than control monocytes, while TS-induced monocyte vacuolization was, at least in part, prevented by Curosurf. Taken together, our data suggest that PS plays a protective role against oxygen radical-mediated, TS-induced cellular stress responses.

Methyl jasmonate induces lauric acid omega-hydroxylase activity and accumulation of CYP94A1 transcripts but does not affect epoxide hydrolase activities in vicia sativa seedlings

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mM solution of MetJA stimulated omega-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min-1 mg-1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid omega-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

Tobacco smoke induces coordinate activation of HSF and inhibition of NFkappaB in human monocytes: effects on TNFalpha release.

Tobacco smoke (TS) exposure is a major risk factor for human disease, and macrophages of healthy smokers have a depressed capacity to release cytokines, including tumor necrosis factor (TNF)alpha. TS induces the synthesis of heat shock (HS)/stress proteins (HSP), and, in particular, of Hsp70. We determined whether Hsp70 induction by TS was mediated by the activation of the HS transcription factor, HSF. HSF activation has been shown to inhibit NFkappaB. Thus, we also determined the effects of TS on NFkappaB. U937 cells and human peripheral blood monocytes were exposed to TS, binding activities of the respective transcription factors were analyzed, and Hsp70 expression and TNFalpha release were determined in parallel. TS activated HSF, which was associated with Hsp70 overexpression and inhibition of NFkappaB binding activity and TNFalpha release. The altered cytokine profile observed in smokers may relate to an HSF/Hsp70-mediated inhibition of NFkappaB activity.

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes.

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.

Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis.

The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.

Cloning, expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that catalyzes in-chain hydroxylation of fatty acids.

Several omega and in-chain fatty acid hydroxylases have been characterized in higher plants. In microsomes from Helianthus tuberosus tuber the omega-2, omega-3, and omega-4 hydroxylation of lauric acid is catalyzed by one or a few closely related aminopyrine- and MnCl2-inducible cytochrome P450(s). To isolate the cDNA and determine the sequences of the(se) enzyme(s), we used antibodies directed against a P450-enriched fraction purified from Mn2+-induced tissues. Screening of a cDNA expression library from aminopyrine-treated tubers led to the identification of a cDNA (CYP81B1) corresponding to a transcript induced by aminopyrine. CYP81B1 was expressed in yeast. A systematic exploration of its function revealed that it specifically catalyzes the hydroxylation of medium chain saturated fatty acids, capric (C10:0), lauric (C12:0), and myristic (C14:0) acids. The same metabolites were obtained with transgenic yeast and plant microsomes, a mixture of omega-1 to omega-5 monohydroxylated products. The three fatty acids were metabolized with high and similar efficiencies, the major position of attack depending on chain length. When lauric acid was the substrate, turnover was 30.7 +/- 1.4 min-1 and Km(app) 788 +/- 400 nM. No metabolism of long chain fatty acids, aromatic molecules, or herbicides was detected. This new fatty acid hydroxylase is typical from higher plants and differs from those already isolated from other living organisms.

Induction of stress proteins by tobacco smoke in human monocytes: modulation by antioxidants.

Tobacco smoke (TS) induced in human monocytes the synthesis of both the classical heat shock proteins (HSP) (Hsp70, Hsp90, Hsp110) and the oxidation-specific stress protein (SP) heme oxygenase (HO). To determine the role of reactive oxygen species in SP induction by TS, we incubated the monocytes with various antioxidants before exposure to TS. Quercetin and N-acetylcysteine (NAC) both prevented the induction of HO by TS but not, or less so, than that of the classical HSP, while the nitric oxide synthase inhibitor L-nitroarginine had no effect. Thus, at least two mechanisms appear involved in SP induction by TS; (i) the induction of HO (oxidation-dependent), which was prevented by quercetin and NAC; and (ii) the induction of Hsp70, which was, at least in part, oxidation-independent. SP induction might represent an adequate biosensor for TS and other radical-mediated environmental exposures.

Characterization of epoxide hydrolase activity in Alternaria alternata f. sp. lycopersici. Possible involvement in toxin production.

Using trans-diphenylpropane oxide (tDPPO) as a substrate, we measured epoxide hydrolase (EH) activity in subcellular fractions of Alternaria alternata f. sp. lycopersici (Aal), a fungus that produces host-specific toxins. The activity was mainly (> 99.5%) located in the soluble fraction (100,000 x g supernatant) with the optimum pH at 7.4. An increase of toxin production between days 3 and 9 found in a Aal liquid culture over a 15 days period was concomitant with a period of high EH activity. EH activity remained constant during the same period in an Alternaria alternata culture, a fungus which does not produce toxin. In vivo treatment of Aal culture with the peroxisome proliferator clofibrate stimulated EH activity by 83% and enhanced toxin production 6.3 fold. Both 4-fluorochalcone oxide (4-FCO) and (2S,3S)-(-)-3-(4-nitrophenyl)-glycidol (SS-NPG) inhibited EH activity in vitro with a I50 of 23 +/- 1 microM and 72 +/- 19 microM, respectively. The possible physiological substrate 9,10-epoxystearic acid was hydrolyzed more efficiently by Aal sEH than the model substrates trans- and cis-stilbene oxide (TSO and CSO) and trans- and cis-diphenylpropane oxide (tDPPO and cDPPO).