RESUMEN
The sensitive differential pulse anodic stripping voltammetry (DPASV) proposed originally by Ishiyama et al. (2001) has been revised and improved to allow the accurate measurement of silicon on a hanging mercury drop electrode (HMDE) instead of a glassy carbon electrode. We assessed the rate of formation of the partially reduced ß-silicododecamolybdate and found that metallic mercury promotes the reaction in the presence of a large concentration of Fe(3+). The scope of the method has been broadened by carrying out the measurements in the presence of a constant amount of Fe(3+). The limit of detection (LOD) of the method described in the present paper is 100 µg Sig(-1) of steel, with a relative precision ranging from 5% to 12%. It can be further enhanced to 700 ng Sig(-1) of steel provided the weight of the sample, the dilution factors, the duration of the electrolysis and the ballast of iron are adequately revised. The tolerance to several interfering species has been examined, especially regarding Al(3+), Cr(3+) and Cr VI species. The method was validated using four low-alloy ferritic steels certified by the National Institute of Standards and Technology (NIST). Its application to nickel base alloys as well as to less complicated matrixes is straightforward. It has also been successfully applied to the determination of free silicon into silicon carbide nano-powder.
Asunto(s)
Técnicas Electroquímicas , Mercurio/química , Silicio/análisis , Acero/química , Acetona/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Compuestos Férricos/química , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Límite de Detección , Molibdeno/química , Compuestos de Silicona/química , SolucionesRESUMEN
Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol-4alpha-methyl oxidase (SMO), a membrane-bound non-heme di-iron oxygenase involved in lipid metabolism. Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis. These results represent the first functional identification of SMO genes from plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Oxigenasas de Función Mixta/genética , Fitosteroles/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/metabolismo , Cromatografía de Gases , Clonación Molecular , ADN Complementario , Ergosterol/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/clasificación , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Oxidación-Reducción , Filogenia , Fitosteroles/química , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.
Asunto(s)
Carboxiliasas/genética , Carboxiliasas/metabolismo , Metabolismo de los Lípidos , Saccharomyces cerevisiae/enzimología , Metanosulfonato de Etilo , Glicéridos/metabolismo , Cinética , Mutagénesis , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Esfingolípidos/metabolismo , TemperaturaRESUMEN
Deuterium-labeled 5alpha-cholest-7-en-3beta-ol (1) bearing one or two deuteriums at the C-5alpha and (or) C-6alpha positions was synthesized in high isotopic and chiral purity. These compounds were used as substrates with the microsomal wild-type Zea mays and recombinant Arabidopsis thaliana Delta(7)-sterol-C5(6)-desaturases (5-DES) to probe directly the stereochemistry and the mechanism of the enzymatic reaction. Clearly, in the conversion of 1 by both 5-DESs, the 6alpha-hydrogen is removed. [6alpha-(2)H]-5alpha-Cholest-7-en-3beta-ol shows an intermolecular deuterium kinetic isotope effect (DKIE) on V and V/K, (D6)V = 2.6+/-0.3, (D6)V/K = 2.4+/-0.1; and (D6)V = 2.3 +/-0.3, (D6)V/K = 2.3+/-0.2 for the Zea mays and A. thaliana wild-type 5-DES, respectively. In contrast, negligible or minor isotope effects, (D5)V = 0.99+/-0.04, (D5)V/K = 0.91+/-0.08; and (D5)V = 0.93 +/-0.06, (D5)V/K = 0.96+/-0.04, respectively, were observed with [5alpha-(2)H]-cholest-7-en-3beta-ol. The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemically activated C6alpha-H bond, a step which appears to be partially rate-limiting in the desaturation process. Cleavage of the C5-H bond has a negligible isotope effect, indicating that the desaturation involves asynchronous scission of the two C-H bonds at C5 and C6. We showed previously [Taton, M., et al. (2000) Biochemistry 39, 701] that threonine 114 was not essential to maintaining desaturase activity, although V/K values for mutant T114I and T114S were respectively 10-fold lower and 4-fold higher than that of the native 5-DES. In this study, we combined variation in enzyme structure and DKIE studies and showed that (D6)V and (D6)V/K increased respectively to 3.8+/-0.3 and 3.8+/-0.4 in mutant T114I and decreased respectively to 1.6+/-0.4 and 1.7+/- 0.1 in mutant T114S. The data suggest that the conserved hydroxyl function at position 114 in the ERG3 family makes the abstraction of the 6alpha-hydrogen atom substantially less rate-limiting during the 5-DES reaction. Based on the data, a tentative mechanism for the desaturation of cholest-7-en-3beta-ol is proposed.
Asunto(s)
Deshidrocolesteroles/química , Deuterio/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/química , Oxidorreductasas/genética , Arabidopsis/enzimología , Arabidopsis/genética , Catálisis , Deshidrocolesteroles/síntesis química , Isoenzimas/química , Isoenzimas/genética , Cinética , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/enzimología , Estereoisomerismo , Especificidad por Sustrato/genética , Zea mays/enzimologíaRESUMEN
The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES. The mutated desaturases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols. In addition, the activities of the recombinant 5-DESs were examined directly in vitro in the corresponding yeast microsomal preparations. One group of mutants was affected in the eight evolutionarily conserved histidine residues from three histidine-rich motifs. Replacement of these residues by leucine or glutamic acid completely eliminated the desaturase activity both in vivo and in vitro, in contrast to mutations at seven other conserved residues. Thus, mutants H203L, H222L, H222E, P201A, G234A, and G234D had a 5-DES activity reduced to 2-20% of the wild-type enzyme, while mutants K115L, P175V, and P175A had a 5-DES activity and catalytical efficiency (V/K) that was similar to that of the wild-type. Therefore, these residues are not essential for the catalysis but contribute to the activity through conformational or other effects. One possible function for the histidine-rich motifs would be to provide the ligands for a presumed catalytic Fe center, as previously proposed for a number of integral membrane enzymes catalyzing desaturations and hydroxylations [Shanklin et al. (1994) Biochemistry 33, 12787-12794]. Another group of mutants was affected in residue 114 based on previous in vivo observations in A. thaliana indicating that mutant T114I was deficient in 5-DES activity. We show that the enzyme T114I has an 8-fold higher Km and 10-fold reduced catalytic efficiency. Conversely, the functionally conservative substituted mutant enzyme T114S displays a 28-fold higher Vmax value and an 8-fold higher Km value than the wild-type enzyme. Consequently, V/K for T114S was 38-fold higher than that for T114I. The data suggest that Thr 114 is involved in stabilization of the enzyme-substrate complex with a marked discrimination between the ground-state and the transition state of a rate-controlling step in the catalysis by the 5-DES.
Asunto(s)
Secuencia Conservada , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/química , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arabidopsis/enzimología , Arabidopsis/genética , Catálisis , Genes de Plantas , Vectores Genéticos/síntesis química , Glicina/genética , Histidina/genética , Humanos , Lisina/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Prolina/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Treonina/genéticaRESUMEN
This report describes recent work on the process of desaturation at C5(6) of sterol precursors in plants. Biochemical characterization of the plant Delta(7)-sterol C5(6)-desaturase (5-DES) indicates that the enzyme system involved shows important similarities to the soluble and membrane-bound non-haem iron desaturases found in eukaryotes, including cyanide and hydrophobic chelators sensitivity, CO resistance and a requirement for exogenous reductant and molecular oxygen. Site-directed mutational analysis of highly conserved residues in 5-DES indicated that eight histidine residues from three histidine-rich motifs were essential for the catalysis, possibly by providing the ligands for a putative Fe centre. This mutational analysis also revealed the catalytic role of the functionally conserved Thr-114.
Asunto(s)
Arabidopsis/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Zea mays/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Catálisis , Secuencia Conservada , Histidina , Humanos , Hierro/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidorreductasas/genética , Plantas Tóxicas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Nicotiana/enzimologíaRESUMEN
A microsomal preparation from seedlings of Zea mays catalyzed the NAD+-dependent oxidative decarboxylation of several substrates, including 4alpha-carboxy-cholest-7-en-3beta-ol, synthesized according to a new procedure, giving the first in vitro evidence for this enzymatic activity in a higher plant. A GC assay has been developed to detect the Delta7-cholestenone produced and the kinetic parameters of the microsomal system have been established. 4alpha-Carboxysterol decarboxylation shows an exclusive requirement for an oxidized pyridine nucleotide, with NAD+ being more efficient than NADP+. The decarboxylation reaction is independent of molecular oxygen. 4alpha-Carboxysterol-C3-dehydrogenase/C4-decarboxylase (4alpha-CD) is a microsome-bound protein which can be efficiently solubilized by detergents, including Brij W-1 and sodium cholate. The Brij W-1-solubilized enzyme was partially purified 290-fold by a combination of DEAE anion-exchange chromatography, Cibacron blue 3GA-agarose dye chromatography, and gel permeation. The apparent molecular mass of 4alpha-CD in sodium cholate was estimated to be 45 kDa. These results support the contention that demethylation at C4 of plant sterols is composed of two separate processes: an oxygen- and NAD(P)H-dependent oxidation of the 4alpha-methyl group to produce the 4alpha-carboxysterol metabolite (S. Pascal et al., J. Biol. Chem. 268, 11639, 1993) followed by oxygen-independent dehydrogenation/decarboxylation to produce an obligatory 3-ketosteroid.
Asunto(s)
Carboxiliasas/química , Carboxiliasas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Zea mays/enzimología , Descarboxilación , Indicadores y Reactivos , Microsomas/enzimología , NAD/química , NADP/química , Solubilidad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Prueba de Complementación Genética , Oxidorreductasas/genética , Triticum/enzimología , Secuencia de Aminoácidos , Azoles/metabolismo , Azoles/farmacología , Secuencia de Bases , Catálisis , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Cartilla de ADN , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Hemo/metabolismo , Datos de Secuencia Molecular , Mutación , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Esterol 14-Desmetilasa , Esteroles/metabolismo , Especificidad por SustratoRESUMEN
SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma1 receptor and emopamil-binding protein, have been characterised molecularly. Only the sigma1 receptor has been shown to bind (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)pro pen-2-ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T-cell proliferation was consistent with a sigma1 receptor-mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil-binding protein is a new SR31747A-binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a delta8-cholesterol isomer at the expense of cholesterol in SR31747A-treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the delta8-delta7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil-binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti-proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol-isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.
Asunto(s)
División Celular/efectos de los fármacos , Colesterol/metabolismo , Ciclohexanos/farmacología , Receptores Opioides , Receptores sigma/metabolismo , Esteroide Isomerasas/antagonistas & inhibidores , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/metabolismo , Humanos , Ratones , Unión Proteica , Receptor Sigma-1RESUMEN
The electron donors for the membrane-bound sterol-4alpha-methyl-oxidases and sterol C5(6)-desaturase of plant sterol biosynthesis have not been previously identified. The requirement of cytochrome b5 to shuttle reducing equivalents from NAD(P)H to 4,4-dimethylsterol-4alpha-methyl oxidase (4,4-DMSO), 4alpha-methylsterol-4alpha-methyl oxidase (4alpha-MSO), and delta7-sterol-C5(6) desaturase (5-DES) was investigated using a purified preparation of IgG raised against plant cytochrome b5. The activities of 4,4-DMSO, 4alpha-MSO, and 5-DES, three oxidative reactions not mediated by cytochrome P-450, were strongly and completely inhibited by the antibody in a microsomal preparation from maize. In addition the IgG also inhibited NADH-dependent cytochrome c reduction in the same preparation. These results strongly suggest that membrane-bound cytochrome b5 of maize microsomes is an obligatory electron carrier from NAD(P)H to 4,4-DMSO, 4alpha-MSO, and 5-DES.
Asunto(s)
Citocromos b5/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas/metabolismo , Esteroles/metabolismo , Zea mays/metabolismo , Metilación , Microsomas/enzimología , NADH Deshidrogenasa/metabolismo , Oxidación-Reducción , Zea mays/enzimologíaRESUMEN
Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae. Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Evolución Molecular , Oxidorreductasas/biosíntesis , Filogenia , Triticum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Colestadienoles/metabolismo , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Cartilla de ADN , ADN Complementario , Biblioteca de Genes , Lanosterol/metabolismo , Microsomas/enzimología , Datos de Secuencia Molecular , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Fitosteroles , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Esterol 14-Desmetilasa , Especificidad por Sustrato , Triticum/genéticaRESUMEN
Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-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 delta8-sterol into delta7-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 delta8-delta7 sterol isomerase are identical proteins in mammals.
Asunto(s)
Proteínas Portadoras/metabolismo , Esteroide Isomerasas/metabolismo , Secuencia de Aminoácidos , Antipsicóticos/metabolismo , Antipsicóticos/farmacología , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/química , Cromatografía de Gases , Cromatografía Líquida de Alta Presión , ADN Complementario/química , Proteínas de Unión al ADN/genética , Haloperidol/metabolismo , Haloperidol/farmacología , Humanos , Datos de Secuencia Molecular , Morfolinas/metabolismo , Morfolinas/farmacología , Sistemas de Lectura Abierta , Unión Proteica , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Esteroles/biosíntesis , Transactivadores/genética , Regulador Transcripcional ERG , Trifluoperazina/metabolismo , Trifluoperazina/farmacologíaRESUMEN
SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.
Asunto(s)
Ciclohexanos/farmacología , Inhibidores Enzimáticos/farmacología , Inmunosupresores/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Esteroide Isomerasas/antagonistas & inhibidores , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Ergosterol/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Esteroide Isomerasas/genética , Transformación GenéticaRESUMEN
A series of mono- and diazasteroids have been synthesized as analogs of a predicted carbocationic intermediate of delta 5,7-sterol delta 7-reductase (delta 7-SR). 6-Aza-B-homo-5 alpha-cholest-7-en-3 beta-ol (4), a novel compound whose synthesis is described for the first time, and 6,7-diaza-5 alpha-cholest-8(14)-en-3 beta-ol (6) were shown to be very powerful inhibitors of delta 7-SR in a preparation isolated from maize (Zea mays) (K(i),app = 50-70 nM, Ki,app/Km,app = 1.0 x 10(-4) to 1.3 x 10(-4). The data are consistent with a carbonium ion mechanism for the reduction; compounds 4 and 6 probably act as reaction intermediate analogs. Compound 4, in contrast to compound 6, displayed in the same microsomal preparation more than 50-fold selectivity for inhibition of the delta 7-SR versus delta 8-delta 7-sterol isomerase, cycloeucalenol isomerase, and delta 8,14-sterol delta 14-reductase, the mechanism of these four enzymes involving presumptive cationic intermediates centered respectively at C7, C8, C9, and C14. These observations highlight the paramount importance of the location of the positively charged nitrogen atom(s) in the B-ring structure for selectivity among these enzymes involving structurally close cationic reaction intermediates. Efficient in vivo inhibition of sterol biosynthesis in bramble cell suspension cultures by a low concentration of compound 4 was demonstrated and confirmed the in vitro properties of this derivative.)
Asunto(s)
Azaesteroides/farmacología , Colesterol/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Fitosteroles/biosíntesis , Zea mays/enzimología , Azaesteroides/síntesis química , Azaesteroides/química , Células Cultivadas , Colesterol/síntesis química , Colesterol/química , Colesterol/farmacología , Espectroscopía de Resonancia Magnética , Microsomas/enzimología , Microsomas/metabolismo , Modelos Químicos , Estructura Molecular , Oxidorreductasas/metabolismo , Fitosteroles/metabolismo , Esteroide Isomerasas/metabolismo , Relación Estructura-Actividad , Zea mays/metabolismoRESUMEN
Microsomes obtained from maize seedlings catalyzed the introduction of the delta5-bond into delta7-sterols to yield the corresponding delta 5,7-sterols. Enzymatic bioassay conditions have been developed for the first time for delta 7-sterol C5(6)-desaturase in photosynthetic organisms. The properties of the microsomal system have been studied and the kinetics of the desaturation reaction has been established. The desaturation reaction requires molecular oxygen and NADH. Coenzyme efficiency studies indicate that NADH is more efficient that NADPH and that in the presence of NADH, NAD+ stimulates the desaturation process but cannot sustain the reaction by itself. The desaturation is strongly inhibited by cyanide, is sensitive to 1,10-phenanthroline and to salicylhydroxamic acid, but is insensitive to carbon monoxide, suggesting the involvement of a metal ion, presumably iron, in an enzyme-bound form in the desaturating system. From a series of incubations with delta 7-sterols and other sterol analogs, the substrate specificity for desaturation was determined. Our data indicate the substrate selectivity of the C5(6)-desaturation for 4-desmethyl-delta 7-sterols. Moreover, the results show that specificity of maize C5(6)-desaturase favored delta 7-sterols possessing a C24-methylene or ethylidene substituent compared to 24-ethyl-substituted delta 7-sterols. Finally, the results demonstrate directly that during plant sterol synthesis the delta 5-bond is introduced via the sequence delta 7-sterol-->delta 5,7-sterol-->delta 5-sterol.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/metabolismo , Plantas/metabolismo , Esteroles/biosíntesis , Coenzimas/metabolismo , Transporte de Electrón , Cinética , Microsomas/metabolismo , Estructura Molecular , Oxidación-Reducción , Oxígeno/metabolismo , Esteroles/química , Especificidad por Sustrato , Zea mays/metabolismoRESUMEN
Microsomes obtained from maize embryos were shown to catalyze the reduction of various sterones to produce stereoselectively the corresponding 3 beta-hydroxy derivatives. Enzymatic assay conditions have been developed to characterize this reduction step and the kinetics of the microsomal system has been established. Sterone reduction shows exclusive dependence on NADPH and is inactive with NADH. It is not sensitive to the azole inhibitors pyrifenox, ketoconazole, and itraconazole nor to phenobarbital nor pyrazole. Based on these coenzyme requirements and inhibitor susceptibility, and according to the common pattern of their classification, the maize microsomal sterone-reducing enzyme belongs to the family of ketone reductases. From a series of incubations with natural or synthetic sterones, the substrate specificity of the reduction at C-3 was determined. Our data indicate particularly that 4 alpha-methyl-9 beta,19-cyclo-C30-sterones and 4-desmethyl-delta 7-C27- or C30-sterones are preferentially reduced, while 4,4-dimethyl-C30- or C31-sterones react poorly. The results support the conclusion that the reductase activity identified is a constitutive component of the microsomal sterol 4-demethylation complex recently identified in photosynthetic organisms (S. Pascal et al., 1993, J. Biol. Chem. 268, 11639). They are consistent with the conclusion that 4 alpha-methylsterones are demethylation products of 4,4-gem-dimethylsterols rather than early intermediates in the 4 alpha-monomethyl-sterols-4-demethylation process.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Cetosteroides/metabolismo , Microsomas/enzimología , Fitosteroles/biosíntesis , Zea mays/enzimología , NADP/metabolismo , Oxidación-Reducción , Especificidad por Sustrato , Zea mays/embriologíaRESUMEN
Membrane-bound enzymatic systems obtained from maize embryos that catalyze the oxidative C4-monodemethylation of 4,4-dimethyl- and 4 alpha-methylsterols have been investigated. Enzymatic assay conditions have been developed for the first time to detect the C4-monodemethylated products formed. The properties of the microsomal systems have been established for co-factor requirements and kinetics. The demethylation process has been interrupted to demonstrate the formation of stable, oxygenated intermediates. In addition to the 3-keto and 3 beta-hydroxy-4-monodemethylated products formed, three new sterols have been identified. 3 beta-Hydroxy-4 beta,14 alpha-dimethyl-5 alpha-ergosta-9 beta,19-cyclo-24(24(1))-en-4 alpha-hydroxy methyl was identified for the first time as the immediate metabolite of 24-methylenecycloartanol by 4 alpha-methyl oxidase in addition to 3 beta-hydroxy-4 beta,14 alpha-dimethyl-5 alpha-ergosta-9 beta,19-cyclo- 24(24(1))-en-4 alpha-carboxylic-acid and 3 beta-hydroxy-5 alpha-stigmasta-7,24(24(1))-dien-4 alpha-carboxylic-acid, intermediates involved respectively in the oxidative demethylation of 24-methylenecycloartanol and 24-ethylidenelophenol. Proton nuclear magnetic resonance studies of enzymatically produced 3 beta-hydroxy-4 beta,14 alpha-dimethyl-5 alpha-ergosta-9 beta,19-cyclo-24(24(1))en-4 alpha-carboxylic acid indicate that the 4 alpha-methyl group of 24-methylenecycloartanol is oxidized and subsequently removed during its enzymatic conversion to cycloeucalenol. From a series of incubations with 25 natural or synthetic 4,4-dimethyl and 4 alpha-methylsterols, a high degree of substrate specificity for the oxidation at C4 of 4,4-dimethyl- and 4 alpha-methylsterols was determined. Our results indicate that oxidation of the 4 alpha-methyl group of the 4,4-geminal dimethylsterols requires the more flexible and presumably bent conformation of 9 beta,19-cyclopropylsterols and the absence of a delta 24(25) unsaturation, whereas the rigid planar conformation of delta 7-unsaturated sterols favors oxidation of 4 alpha-methylsterols. Distinct strict structural requirements for the oxidation of 4,4-dimethyl- and 4 alpha-methylsterols and different sensitivity toward cyanide ions and 3 beta,5 alpha,6 alpha-stigmastatriol, a novel inhibitor of 4 alpha-methylsterol C4 oxidase activity, are consistent with the conclusion that two distinct oxidative systems are involved in the removal of the first and second C4-methyl group of phytosterol precursors. Moreover, the present study directly establishes that during the conversion of cycloartenol to phytosterol one C4 dealkylation occurs before the removal of the 14 alpha-methyl group.
Asunto(s)
Microsomas/enzimología , Oxigenasas de Función Mixta/metabolismo , Esteroles/metabolismo , Zea mays/enzimología , Indicadores y Reactivos , Cinética , Espectrometría de Masas , Estructura Molecular , Esteroles/biosíntesis , Esteroles/síntesis química , Especificidad por Sustrato , TritioRESUMEN
Monocyclic and tricyclic compounds possessing a nitrogen atom situated at a position corresponding to the carbenium ion of high energy intermediates or transition states involved during cyclization of 2,3-oxidosqualene to tetra- and pentacyclic triterpenes have been synthesized. These compounds were tested as inhibitors of 2,3-oxidosqualene cycloartenol, lanosterol-, and beta(alpha)-amyrin-cyclases in vitro and in vivo, and their affinity was compared to that of formerly synthesized 8-aza-bicyclic compounds [Taton et al. (1986) Biochem. Biophys. Res. Commun. 138, 764-770]. A monocyclic N-alkyl-hydroxypiperidine was shown to be the strongest inhibitor of the series upon cycloartenol-cyclase (I50 = 1 microM) from maize embryos but was much less effective on the beta(alpha)-amyrin-cyclases from Rubus fruticosus suspension cultures or pea cotyledons. In contrast, 13-aza-tricyclic derivatives displayed little inhibition on 2,3-oxidosqualene cycloartenol-, lanosterol-, and beta(alpha)-amyrin-cyclases. The obtained data exemplify the differences existing in the cyclization process between cycloartenol- (lanosterol-) cyclases on one hand and beta(alpha)-amyrin-cyclases on the other. The results are discussed with respect to current mechanisms postulated for 2,3-oxidosqualene cyclization. Because of its activity in vivo and in vitro the monocyclic N-alkyl-hydroxypiperidine appears to be a potent and promising tool to study sterol biosynthesis regulation.
Asunto(s)
Transferasas Intramoleculares , Isomerasas/antagonistas & inhibidores , Escualeno/análogos & derivados , Animales , Ciclización , Fabaceae/enzimología , Hígado/enzimología , Estructura Molecular , Plantas Medicinales , Ratas , Escualeno/síntesis química , Escualeno/química , Escualeno/farmacología , Relación Estructura-Actividad , Zea mays/enzimologíaRESUMEN
The R-(-) isomer of methyl 1-(2,2-dimethylindan-1-yl)imidazole-5-carboxylate (CGA 214372; 2) strongly inhibited P450-dependent obtusifoliol 14 alpha-demethylase (P450OBT.14DM) (I50 = 8 x 10(-9) M, I50/Km = 5 x 10(-5) in a maize (Zea mays) microsomal preparation. Kinetic studies indicated uncompetitive inhibition with respect to obtusifoliol. The corresponding S-(+) isomer was a 20-fold weaker inhibitor for P450OBT.14DM. The molecular features of a variety of analogues of 2 were related to their potency as inhibitors of P450OBT.14DM in vitro, allowing delineation of the key structural requirements governing inhibition of the demethylase. CGA 214372 proved to have a high degree of selectivity for P450OBT.14DM. This allowed easy distinction of this activity from other P450-dependent activities present in the maize microsomal preparation and gave strong evidence that P450OBT.14DM is a herbicidal target. Microsomal maize P450OBT.14DM and yeast P450LAN.14DM, the only known examples of P450-dependent enzymes carrying out an identical metabolic function in different eukaryotes, showed distinct inhibition patterns with CGA 214372 and ketoconazole, a substituted imidazole anti-mycotic.
Asunto(s)
Azoles/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Microsomas/enzimología , Oxidorreductasas/antagonistas & inhibidores , Esteroles/metabolismo , Zea mays/enzimología , Cinética , Esterol 14-Desmetilasa , Relación Estructura-ActividadRESUMEN
A series of 7-oxo-obtusifoliol analogues have been synthetized and investigated as potential inhibitors of cytochrome P-450 dependent obtusifoliol 14 alpha-demethylase (P-450OBT.14DM) from higher plant microsomes. 7-Oxo-24 xi(24')-dihydro-obtusifoliol and 7-oxo-24(25)-dihydro-29-nor-lanosterol were potent competitive inhibitors for P-450OBT.14DM, binding 125-200 times more tightly than the substrates obtusifoliol and 24(25)-dihydro-29-nor-lanosterol. Inhibition of P-450OBT.14DM by these analogues showed strict structural requirements including the 8-en-7-one system which was compulsory for binding. 7-Oxo-24(25)-dihydro-lanosterol possessing an additional 4 beta-methyl substituent, did not have such inhibitory effects. Treatment of cultures of suspended bramble cells with 7-oxo-24(25)-dihydro-29-nor-lanosterol resulted in a strong decrease of [14C]acetate incorporation into the demethylsterols fraction and in an accumulation of [14C]obtusifoliol. This confirms that P-450OBT.14DM is the main in vivo target of 7-oxo-24(25)-dihydro-29-nor-lanosterol in the sterol-biosynthetic pathway.