Novel Functional Derivatives of Methyl-cis-9,10-Epoxy-Octadecanoate

The synthesis of functional derivatives of epoxystearic acid methyl ester by oxirane ring opening and transesterification of ester group has been described. Some novel surface-active and peroxide-containing compounds have been obtained. Major features of the process have been investigated and main characteristics have been determined.

Cloning, Functional Expression, and Characterization of CYP709C1, the First Sub-terminal Hydroxylase of Long Chain Fatty Acid in Plants

We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the ω-1 and ω-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. Km(app) of 8 μm and Vmax(app) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed.

Stereochemical features of the hydrolysis of 9,10-epoxystearic acid catalysed by plant and mammalian epoxide hydrolases

cis-9,10-Epoxystearic acid was used as a tool to probe the active sites of epoxide hydrolases (EHs) of mammalian and plant origin. We have compared the stereochemical features of the hydrolysis of this substrate catalysed by soluble and membrane-bound rat liver EHs, by soluble EH (purified to apparent homogeneity) obtained from maize seedlings or celeriac roots, and by recombinant soybean EH expressed in yeast. Plant EHs were found to differ in their enantioselectivity, i.e. their ability to discriminate between the two enantiomers of 9,10-epoxystearic acid. For example, while the maize enzyme hydrated both enantiomers at the same rate, the EH from soybean exhibited very high enantioselectivity in favour of 9R,10S-epoxystearic acid. This latter enzyme also exhibited a strict stereoselectivity, i.e. it hydrolysed the racemic substrate with a very high enantioconvergence, yielding a single chiral diol product, threo-9R,10R-dihydroxystearic acid. Soybean EH shared these distinctive stereochemical features with the membrane-bound rat liver EH. The stereochemical outcome of these enzymes probably results from a stereoselective attack by the nucleophilic residue on the oxirane ring carbon having the (S)-configuration, leading to the presumed (in plant EH) covalent acyl–enzyme intermediate. In sharp contrast, the reactions catalysed by cytosolic rat liver EH exhibited a complete absence of enantioselectivity and enantioconvergence; this latter effect might be ascribed to a regioselective formation of the acyl–enzyme intermediate involving C-10 of 9,10-epoxystearic acid, independent of its configuration. Thus, compared with soybean EH, the active site of rat liver soluble EH displays a very distinct means of anchoring the oxirane ring of the fatty acid epoxides, and therefore appears to be a poor model for mapping the catalytic domain of plant EHs.

ω-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94AI: possible involvement in plant defence

The C18 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 Km and Vmax values of 1.2±0.1 μM and 19.2±0.3 nmol/min per nmol of cytochrome P450 for the 9R, 10S enantiomer and of 5.9±0.1 μM 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 C16 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 C18 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 Vmax/Km 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 ω-hydroxylase, with a measured Vmax/Km 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 Vmax/Km values for epoxide ≫ oleic acid > diol.

The Lipids of Pneumocystis carinii

SUMMARY Information about a number of Pneumocystis carinii lipids obtained by the analyses of organisms isolated and purified from infected lungs of corticosteroid-immunosuppressed rats has been reported in recent years. Of the common opportunistic protists associated with AIDS (Cryptosporidium, Toxoplasma, and the microsporidia), more is currently known about the lipids of P. carinii than the others. Lipids that are synthesized by the organism but not by humans are attractive targets for drug development. Thus, the elucidation of Δ7C-24-alykylated sterol and cis-9,10-epoxystearic acid biosyntheses in P. carinii is currently being examined in detail, since these have been identified as P. carinii-specific lipids. The development of low-toxicity drugs that prevent sterol C-24 alkylation and the specific inhibition of the lipoxygenase that forms cis-9,10-epoxystearic acid might prove fruitful. Although humans can synthesize coenzyme Q10, the anti-P. carinii activity and low toxicity of ubiquinone analogs such as atovaquone suggest that the electron transport chain in the pathogen may differ importantly from that in the host. Although resistance to atovaquone has been observed, development of other naphthoquinone drugs would provide a broader armamentarium of drugs to treat patients with P. carinii pneumonia. Studies of bronchoalveolar lavage fluid and of infected lungs have demonstrated that the infection causes a number of chemical abnormalities. Bronchoalveolar lavage fluid obtained after the removal of lung cellular material and the organisms has been shown to contain larger amounts of surfactant proteins and smaller amounts of phospholipids than do comparable samples from P. carinii-free lungs. Increased phospholipase activity, inhibition of surfactant secretion by type II cells, and uptake and catabolism of lipids by the pathogen may explain this phenomenon related to P. carinii pneumonia. Although not yet thoroughly examined, initial studies on the uptake and metabolism of lipids by P. carinii suggest that the organism relies heavily on exogenous lipid nutrients.