Maryse Taton

Emopamil-binding Protein, a Mammalian Protein That Binds a Series of Structurally Diverse Neuroprotective Agents, Exhibits Δ8-Δ7 Sterol Isomerase Activity in Yeast

Δ8-Δ7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of Δ8-sterols to their corresponding Δ7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert Δ8-sterol into Δ7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and Δ8-Δ7 sterol isomerase are identical proteins in mammals.

Interaction of triazole fungicides and plant growth regulators with microsomal cytochrome P-450-dependent obtusifoliol 14α-methyl demethylase

Abstract A number of triazole fungicides and plant growth regulators (PGRs) were applied to maize seedlings in vivo and total sterol composition was analyzed, quantified, and for the first time assayed on the plant microsomal obtusifoliol 14α-methyl demethylase ( P -450 OBT.14DM ) from maize embryos, which has been previously shown to be a cytochrome (cyt) P -450-dependent monoxygenase (A. Rahier and M. Taton, Biochem. Biophys. Res. Commun. 140 , 1064–1072 1986. The in vivo effect of such compounds was modification of the sterol profile leading to a more or less pronounced accumulation of 14α-methyl sterols, (obtusifoliol, 24-dihydro-obtusifoliol, 14α-methyl-fecosterol) at the expense of Δ 5 -sterols present in the control; (campesterol, stigmasterol, and sitosterol). All the compounds inhibited P -450 OBT.14DM in vitro and the IC 50 for the different triazoles were measured and shown to be in the range 0.05–10 μ M . Comparison of the in vivo and in vitro data showed a good correlation in accord with P -450 OBT.14DM being the target of these fungicides and gave the following orders of potency: in vivo , Lab 170250F > Lab 158241F > propiconazole > BAS 111 > BAS 110 = triadimefon ⪢ tetcyclacis and in vitro , Lab 170250F ⪢ Lab 158241F ⪢ propiconazole = BAS 111 > triadimefon = BAS 110 = tetcyclacis. Moreover, most of fungicides, Lab 170250F, Lab 158241F, propiconazole, were shown to inhibit P -450 OBT.14DM more strongly than the PGRs, BAS 110, BAS 111, and tetcyclacis, and also to modify the sterol profile of treated plants more efficiently. Lab 170250F, propiconazole, and tetcyclacis were shown to induce Type II spectral changes in accord with the binding of these compounds to cyt P -450, the measured K d values 20–30 μ M differing strongly from the IC 50 values on the P -450 OBT.14DM activity. Moreover this binding with the total cyt P -450 contained in maize microsomes matched neither the IC 50 values nor the fungicidal activity.

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ8-Δ7-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10−8 M, Ki/Km=10−3) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.

Inhibition of δ8 → δ7-sterol isomerase and of cycloeucalenol—obtusifoliol isomerase by N-benzyl-8-aza-4α, 10-dimethyl-trans-decal-3β-ol, an analogue of a carbocationic high energy intermediate

Abstract An enzymatic assay for the δ 8 → δ 7 -sterol isomerase, an enzyme involved in sterol biosynthesis, has been developed in higher plants. This assay has been used in the study of various inhibitors. N -Benzyl-8-aza-4α, 10-dimethyl- trans -decal-3β-ol was designed to mimic the C-8 and the C-9 carbocationic high energy intermediates occurring during the reactions catalysed by the δ 8 → δ 7 -sterol isomerase and the cycloeucalenol obtusifoliol isomerase, respectively. In accordance with the ‘transition state analogues’ theory, this analogue of a high energy intermediate was found to be a very potent and specific inhibitor of the two enzymatic reactions both in vitro and in vivo .

The 14α-demethylation of obtusifoliol by a cytochrome P-450 monooxygenase from higher plants microsomes

Microsomes isolated from corn embryos (Zea mays) can demethylate the 14 alpha-methyl group of obtusifoliol 2. An enzymatic assay has been developed for obtusifoliol 14 alpha-methyl-demethylase in higher plants. The enzymatic reaction was shown to occur sequentially, converting obtusifoliol 2 to 4 alpha-methyl-5 alpha-ergosta-8,24(28)-dien-3 beta-ol 4 via the trienol 4 alpha-methyl-5 alpha-ergosta-8,14,24(28)-trien-3 beta-ol 3 which was thoroughly identified. This enzymatic reaction is dependent of NADPH and molecular oxygen. It is inhibited by CO, menadione and specific inhibitors of cytochrome P-450, the CO inhibition being partially reversed by light. It is concluded that in Zea mays microsomes, obtusifoliol is demethylated at C-14 by a cytochrome P-450 containing monooxygenase system.

Optimized expression and catalytic properties of a wheat obtusifoliol 14α-demethylase (CYP51) expressed in yeast

CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14alpha-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C. E. & Halkier, B.A. (1997) Plant J. 11, 191-201] and wheat [Cabello-Hurtado, F., Zimmerlin, A., Rahier, A., Taton, M., DeRose, R., Nedelkina, S., Batard, Y., Durst, F., Pallett, K.E. & Werck-Reichhart, D. (1997) Biophys. Biochem. Res. Commun. 230, 381-385]. Wheat and sorghum CYP51 proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32-39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum-wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450.(L culture)-1, with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14alpha-demethylation of obtusifoliol very actively (Km,app = 197 microm, kcat = 1.2 min-1) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (Ks = 2.2 microM) and eburicol (Ks = 2.5 microm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (Ks = 289 microM), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and gamma-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The Ks of plant CYP51 for LAB170250F (0.29 microM) and gamma-ketotriazole (0.40 microM) calculated from the type-II sp2 nitrogen-binding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.

N-|(1,5,9)-trimethyl-decyl|-4α,10-dimethyl-8-aza-trans-decal-3β-ol, a novel potent inhibitor of 2,3-oxidosqualene cycloartenol and lanosterol cyclases

Summary N-|(1, 5, 9)-trimethyl-decyl)|-4α, 10-dimethyl-8-aza-trans-decal-3β-ol 9 was designed to mimic the C9 or C8 high energy carbocationic intermediates postulated during the enzymic cyclization of 2,3-oxidosqualene to different triterpenes. The structurally new molecule 9 inhibits strongly cycloartenol and lanosterol cyclases in maize seedlings and rat liver microsomes respectively, whereas it does not inhibit β-amyrin cyclase in the plant system. For the first time 2,3-oxidosqualene cycloartenol cyclase and β-amyrin cyclase have been differentiated in the same plant material by use of a specific inhibitor.

Microsomal Δ8,14-sterol Δ14-reductase in higher plants

An enzymatic assay for delta 8,14-sterol delta 14-reductase, an enzyme involved in sterol biosynthesis, has been developed for the first time in higher plants. The properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics and substrate specificity. This enzymatic double-bound reduction is thought to proceed through an electrophilic addition mechanism, involving a C14 putative carbonium ion high-energy intermediate. Using this in vitro assay, ammonium and iminium analogues of this cationic intermediate were shown to be potent inhibitors of the reduction reaction. Thus, compounds of the N-alkyl-8-aza-4 alpha,10-dimethyl-trans-decal-3 beta-ol series strongly inhibited sterol reductase (I50 = 0.07 - 4 microM) (I50/Km = 10(-4) - 10(-3), as did the antimycotic agent 15-azasterol (I50 = 0.03 microM); all of these compounds act as reaction-intermediate analogues of the proposed C14 carbonium ion intermediate. Moreover, the in vitro inhibition of the plant sterol reductase by a series of ammonium-ion-containing fungicides was demonstrated. The relative specificity of these different series of inhibitors toward cycloeucalenol-obtusifoliol isomerase, delta 8----delta 7-sterol isomerase and delta 8,14-sterol delta 14-reductase, was directly studied.

Identification of Δ5,7-sterol-Δ7-reductase in higher plant microsomes

Summary A microsomal preparation from seedlings of Zea mays catalyzed the NADPH dependent reduction of the Δ 7 -bond of Δ 5,7 -cholestadienol (1) giving the first in vitro evidence for the intermediacy of Δ 5,7 -sterols in plant sterol biosynthesis. Using a GC assay developed to detect the cholesterol (2) produced, the properties of the microsomal enzyme have been established with respect to cofactor requirements and kinetics. The potent in vitro inhibition of the plant Δ 5,7 -sterol-Δ 7 -reductase by the ammonium-ion containing fungicides, tridemorph (3), fenpropimorph (4) and AY 9944 (5) was demonstrated. The high affinities observed for these derivatives, especially for (4) (I 50 = 8×10 −8 M, I 50 /K m = 2×10 −4 ), are in full accordance with the previously proposed cationic mechanism involved in this reduction reaction.

Comparative study of the inhibition of sterol biosynthesis in Rubus fruticosus suspension cultures and zea mays seedlings by N-(1,5,9-trimethyldecyl)-4α,10-dimethyl-8-aza-trans-decal-3β-ol and derivatives

Abstract The nitrogen substituents present in tridemorph and fenpropimorph, which are systemic fungicides, have been linked to an 8-aza-bicyclic skeleton leading to N-(1,5,9-trimethyldecyl)-4α,10-dimethyl-8-aza-trans-decal-3β-ol and N-(3-(4-tert-butylphenyl-)2-methyl)-propyl-8-aza-4α,10-dimethyl-trans-decal-3β-ol respectively. The latter two compounds present in a stable molecule key structural elements of unstable C-8 and C-9 carbocationic high-energy intermediates which occur during the reactions catalysed by the Δ8 → Δ7-sterol isomerase and the cycloeucalenol-obtusifoliol isomerase, respectively. When given to either bramble cell suspension cultures or maize seedlings, they led to a spectacular accumulation of 9β,19-cyclopropyl sterols and were in that respect much more efficient than any known molecules and in particular than the N-benzyl decalin previously described which led to accumulation of Δ8-sterols. Surprisingly, treatment of the plant cells by the N-oxide derivatives of the N-benzyl decalin resulted in dramatic accumulation of Δ8,14-sterols.