RESUMEN
Biotin synthase, a member of the 'radical SAM' (S-adenosylmethionine) family, converts DTB (dethiobiotin) into biotin. The active form of the Escherichia coli enzyme contains two (Fe-S) centres, a (4Fe-4S) and a (2Fe-2S). The (4Fe-4S)2+/+ mediates the electron transfer required for the reductive cleavage of SAM into methionine and a DOA* (deoxyadenosyl radical). Two DOA*, i.e. two SAM molecules, are consumed to activate the positions 6 and 9 of DTB. A direct transfer of isotope from the labelled substrate into DOAH (deoxyadenosine) has been observed with 2H, although not quantitatively, but not with tritium. The source of the sulphur introduced to form biotin is still under debate. We have shown that the (2Fe-2S)2+ cluster can be reconstituted in the apoenzyme with S2- and Fe2+. When S2- was replaced by [34S2-], [35S2-] or Se2-, biotin containing mostly the sulphur isotopes or selenium was obtained. This leads us to favour the hypothesis that the (2Fe-2S) centre is the sulphur donor, which may explain the absence of turnover of the enzyme. DTBSH (9-mercaptodethiobiotin), which already contains the sulphur atom of biotin, was shown to be an alternative substrate of biotin synthase both in vivo and with a crude extract. When this compound was tested with a well-defined in vitro system, the same turnover of one and similar reaction rates were observed for DTB and DTBSH. We postulate that the same intermediate is formed from both substrates.
Asunto(s)
Biotina/biosíntesis , Sulfurtransferasas/metabolismo , Biotina/análogos & derivados , Biotina/química , Biotina/metabolismo , Radicales Libres , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Cinética , Azufre/metabolismo , Sulfurtransferasas/químicaRESUMEN
The genetics and mechanistic enzymology of biotin biosynthesis have been the subject of much investigation in the last decade, owing to the interest for biotin production by fermentation, on the one hand, and for the design of inhibitors with potential herbicidal properties, on the other hand. Four enzymes are involved in the synthesis of biotin from its two precursors, alanine and pimeloyl-CoA. They are now well-characterized and the X-ray structures of the first three have been published. 8-Amino-7-oxopelargonic acid synthase is a pyridoxal 5'-phosphate (PLP) enzyme, very similar to other acyl-CoA alpha-oxoamine synthases, and its detailed mechanism has been determined. The origin of its specific substrate, pimeloyl-CoA, however, is not completely established. It could be produced by a modified fatty acid pathway involving a malonyl thioester as the starter. 7,8-Diaminopelargonic acid (DAPA) aminotransferase, although sharing sequence and folding homologies with other transaminases, is unique as it uses S-adenosylmethionine (AdoMet) as the NH2 donor. The mechanism of dethiobiotin synthethase is also now well understood. It catalyzes the formation of the ureido ring via a DAPA carbamate activated with ATP. On the other hand, the mechanism of the last enzyme, biotin synthase, which has long raised a very puzzling problem, is only starting to be unraveled and appears indeed to be very complex. Biotin synthase belongs to the family of AdoMet-dependent enzymes that reductively cleave AdoMet into a deoxyadenosyl radical, and it is responsible for the homolytic cleavage of C-H bonds. A first radical formed on dethiobiotin is trapped by the sulfur donor, which was found to be the iron-sulfur (Fe-S) center contained in the enzyme, and cyclization follows in a second step. Two important features come from these results: (1) a new role for an Fe-S center has been revealed, and (2) biotin synthase is not only a catalyst but also a substrate for the reaction. Lipoate synthase, which catalyzes the formation of two C-S bonds from octanoic acid, has a very high sequence similarity with biotin synthase. Although no in vitro enzymology has been carried out with lipoate synthase, the sequence homology as well as the results of in vivo studies support the conclusion that both enzymes are strongly mechanistically related.
Asunto(s)
Biotina/biosíntesis , Escherichia coli/genética , Ácido Tióctico/biosíntesis , Acilcoenzima A/metabolismo , Bacillus/enzimología , Bacillus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Herbicidas/metabolismo , Cinética , MutaciónRESUMEN
We previously showed that biotin synthase in which the (Fe-S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe-2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.
Asunto(s)
Biotina/metabolismo , Cisteína/metabolismo , Escherichia coli/enzimología , Proteínas Hierro-Azufre/metabolismo , Sulfuros/metabolismo , Sulfurtransferasas/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
The nature and properties of the iron-sulphur (Fe-S) cluster in as-prepared and reduced biotin synthase of Escherichia coli have been investigated by Mössbauer spectroscopy. Our data clearly demonstrate that in the as-prepared sample, the cluster is present as [2Fe-2S](2+) with isomer shift, delta = 0.29 mm/s and quadrupole splitting, DeltaE(Q) = 0.53 mm/s, indicating incomplete cysteinyl-S coordination. Anaerobic reduction by dithionite in the presence of 55% (v/v) glycerol converts this form to [4Fe-4S](2+) (delta = 0.45 mm/s and DeltaE(Q) = 1.11 mm/s) and is accompanied by some destruction to Fe(2+). This cluster conversion is reversible and when exposed to air, the [4Fe-4S](2+) cluster is quantitatively reconverted to the [2Fe-2S](2+) cluster without any further cluster degradation.
Asunto(s)
Escherichia coli/enzimología , Hierro/química , Sulfurtransferasas/metabolismo , Aire , Proteínas Hierro-Azufre/metabolismo , Espectroscopía de Mossbauer , Sulfurtransferasas/químicaRESUMEN
Biotin synthase catalyses the last step of the biosynthesis of biotin in microorganisms and plants. The active protein isolated from Bacillus sphaericus and Escherichia coli contains an iron-sulphur (FeS) cluster. The native enzymes were depleted of their iron and inorganic sulphide and the resulting apoenzymes were chemically reconstituted with FeCl3 and Na2[34S] to give labelled (Fe34S) enzymes. These enzymes were functional and when assayed in vitro produced labelled biotin containing about 65% of 34S. These data strongly support the hypothesis that the sulphur of biotin is derived from the (FeS) centre of the enzyme.
Asunto(s)
Bacillus/enzimología , Biotina/biosíntesis , Escherichia coli/enzimología , Azufre/metabolismo , Sulfurtransferasas/metabolismo , Apoenzimas/metabolismo , Bacillus/metabolismo , Proteínas Bacterianas/metabolismo , Biotina/química , Cloruros , Escherichia coli/metabolismo , Compuestos Férricos/metabolismo , Holoenzimas/metabolismo , Hierro/metabolismo , Cinética , Espectrometría de Masas , Modelos Químicos , Sulfuros/metabolismo , Sulfurtransferasas/químicaRESUMEN
The fact that biotin synthase, from Escherichia coli and Bacillus sphaericus, requires S-adenosylmethionine and a reducing system led us to postulate that this synthase could belong to the family of enzymes which use S-adenosylmethionine as a source of deoxyadenosyl radical, namely pyruvate formate-lyase, lysine 2,3-aminomutase, and anaerobic ribonucleotide reductase. We describe here experiments with S-[2,8-(3)H] adenosylmethionine and S-adenosyl-[methyl-3H]methionine which allowed the identification and quantification of the expected cleavage products, deoxyadenosine, and methionine. They are formed in equimolar amounts, in a ratio close to 3 with respect to the biotin produced. We postulate a mechanism involving the homolytic cleavage of two C-H bonds which should consume two equivalents of S-adenosylmethionine. The observed excess of S-adenosylmethionine consumption is attributed to abortive processes.
Asunto(s)
Bacillus/enzimología , Biotina/biosíntesis , Escherichia coli/enzimología , S-Adenosilmetionina/metabolismo , Sulfurtransferasas/metabolismo , Radicales Libres , S-Adenosilmetionina/químicaRESUMEN
Radical species are postulated intermediates in the formation of the carbon-sulfur bonds of biotin. It was of interest to examine the behaviour of unsaturated analogues which should give rise to allylic radicals. The two isomers of 4,5-dehydrodethiobiotin have been synthesized and labelled with 14C on their carboxylic acid group. When incubated with an in vitro system capable of transforming dethiobiotin into biotin, they covalently label biotin synthase.
Asunto(s)
Imidazoles/síntesis química , Biotina/análogos & derivados , Biotina/metabolismo , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Imidazoles/química , Sulfurtransferasas/metabolismoRESUMEN
Biotin synthase from Bacillus sphaericus has been purified to homogeneity from a recombinant strain. The UV-visible spectrum of the pure protein reveals the presence of a [2Fe-2S] cluster. The enzyme is active in the conversion of dethiobiotin to biotin in vitro, in the presence of NADPH, AdoMet and additional unidentified components from the crude extract of B. sphaericus wild type. We have also found that photoreduced deazaflavin can substitute for the crude extract and NADPH. In this system, biotin synthase is capable of transforming dethiobiotin into biotin in the absence of any other protein but at a substoichiometric level. When this assay was conducted in the presence of [35S]cysteine, no 35S was incorporated into biotin, contrary to what happens in the presence of the crude extract.
Asunto(s)
Biotina/análogos & derivados , Biotina/metabolismo , Sulfurtransferasas/metabolismo , Secuencia de Aminoácidos , Bacillus/enzimología , Sistema Libre de Células , Cisteína/metabolismo , Flavinas/metabolismo , Datos de Secuencia Molecular , Análisis Espectral , Azufre/metabolismo , Sulfurtransferasas/aislamiento & purificaciónRESUMEN
The activity of biotin synthesis from dethiobiotin was found in cell-free extracts of an Escherichia coli bioB transformant. Among the sulfur compounds tested, only S-adenosyl-L-methionine (AdoMet) had a significant effect, while methionine and cysteine were inert. The activity was linearly stimulated by increasing protein concentration. When the dialyzed cell-free extracts were used for the reaction, NADP+, NADPH, and FAD among the well-known cofactors tested promoted the activity. Furthermore, in the presence of AdoMet, cysteine was apparently effective for biotin synthetic activity.
Asunto(s)
Biotina/análogos & derivados , Biotina/biosíntesis , Cisteína/farmacología , Escherichia coli/enzimología , S-Adenosilmetionina/farmacología , Bacillus/enzimología , Bacillus/metabolismo , Biotina/metabolismo , Extractos Celulares/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Flavina-Adenina Dinucleótido/farmacología , Inmunosupresores , NAD/metabolismo , NAD/farmacología , NADP/metabolismo , NADP/farmacología , S-Adenosilmetionina/metabolismoRESUMEN
Tracheobronchial vasoconstriction and subsequent reduction of airway wall thickness due to the alpha 1-agonist methoxamine, might be responsible for prevention of exercise-induced asthma, and reduction of bronchial hyperresponsiveness to methacholine increase in exercise performance in patients with impaired left ventricular function. Since bronchial wall oedema plays an important role in asthma, we have now investigated the bronchial response to the intravenously administered alpha 1-agonist, phenylephrine, in asthma of various severity. Increasing noncumulative intravenous phenylephrine doses (100 to 600 micrograms) were injected in 18 asthmatic subjects (three groups: mild asthma, mild asthma with recent acute attack, severe obstructive asthma) and in 11 control subjects. Changes in specific airways resistance (sRaw) on phenylephrine were linearly related to the dose administered in 16 out of 18 asthmatic subjects, and in only 3 out of 11 control subjects. In the asthmatic subjects, sRaw increased in 10 patients whose asthma was mild, or bronchial obstruction mild to moderate, and decreased in the remaining 8 asthmatic subjects with more severe disease or with a higher degree of bronchial obstruction. Changes in forced expiratory volume in one second (FEV1) were consistent with those of sRaw. In the five asthmatic subjects who underwent the protocol twice, results were reproducible. There was no difference in the responses of heart rate between the three groups of asthmatic subjects. It is likely that phenylephrine acts both via airway smooth muscle contraction, an effect which might predominate in mild asthma, and via mucosal vasoconstriction, which might overcome the effect on smooth muscle in more severe asthma with bronchial wall oedema.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Resistencia de las Vías Respiratorias/efectos de los fármacos , Asma/fisiopatología , Hiperreactividad Bronquial/fisiopatología , Broncoconstricción/efectos de los fármacos , Fenilefrina/farmacología , Adulto , Broncoconstricción/fisiología , Estudios de Casos y Controles , Relación Dosis-Respuesta a Droga , Femenino , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Infusiones Intravenosas , Masculino , Fenilefrina/administración & dosificación , Reproducibilidad de los Resultados , Vasoconstricción/fisiologíaRESUMEN
The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant. Among the sulfur compounds tested, only S-adenosyl-L-methionine was active, while L-methionine and L-cysteine had no significant effect. Protein concentrations higher than 15 mg/ml in the reaction mixture were needed to detect biotin synthase activity. When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe2+, Mn2+, and Ca2+ among the metal ions tested also had some effects.
Asunto(s)
Bacillus/enzimología , Bacillus/genética , Biotina/análogos & derivados , Biotina/metabolismo , Sulfurtransferasas/metabolismo , Transformación Bacteriana , Proteínas Bacterianas/metabolismo , Sistema Libre de Células , Coenzimas/metabolismo , Coenzimas/farmacología , Cinética , Metales/farmacología , Azufre/farmacologíaRESUMEN
A cell-free system of a bioB transformant of Bacillus sphaericus, effecting the last step of biotin biosynthesis, namely the introduction of sulfur into dethiobiotin has been recently described. S-adenosyl methionine (SAM) is absolutely necessary for activity. We show here, through experiments with [35S]SAM and [35S]Cys, that the sulfur donor is not SAM but probably cysteine (Cys) or a derivative. This finding together with the fact that NADPH and FAD are required for activity leads us to postulate some analogy between the biotin synthase system and other systems which use SAM as a source of desoxyadenosyl radical.
Asunto(s)
Bacillus/enzimología , Biotina/biosíntesis , Sulfurtransferasas/metabolismo , Cisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Laboratory and field experiments were conducted to determine the best procedure for predicting the discomfort caused by multi-axis vibration. In the laboratory experiment, 11 seated subjects compared single-axis vibration in one axis to single-axis vibration in another axis, and compared dual-axis vibration to single-axis vibration. In the field experiment, 22 lorry drivers rated the discomfort of 16 different rides. The results show that the best procedure for predicting the discomfort is to combine the vibration inputs by taking the square root of the sum of squares of the weighted r.m.s. values of the vibration in each axis.
Asunto(s)
Ergonomía/métodos , Vibración , Adulto , Conducción de Automóvil , Humanos , Masculino , Modelos Teóricos , Valor Predictivo de las Pruebas , Vibración/efectos adversosRESUMEN
It is still debated as to whether the bronchospasm induced by hyperpnoea in asthmatic subjects is followed by a period of refractoriness to a subsequent challenge. We studied, therefore, the effect of repeated challenges with eucapnic hyperpnoea in asthmatic subjects and compared it to that in normal subjects. Ten normal and 34 asthmatic subjects were challenged twice with a steady isocapnic hyperventilation (25 l X min-1 X m-2 BSA for 6 min) of dry air at room temperature. The interval between challenges was 30 min in the normal subjects and was 30-60 min in asthmatic subjects to allow for full recovery of FEV1 before the second challenge. In the normal subjects, neither the first nor the second challenge caused a detectable change in FEV1. In the asthmatic subjects, the fall in FEV1 was on average less marked at 5, 8 and 10 min after the second challenge than after the first one (p less than 0.05 by analysis of variance). Analysis of data from individuals showed partial to full refractoriness in 14 of the 34 subjects. In no instance was the fall in FEV1 significantly greater after the second challenge than after the first one. Thirteen other asthmatic subjects were challenged twice at a 30-60 min interval with stepwise increases in ventilation of dry air at room temperature until wheezing or chest tightness occurred or a ventilation of 50 l X min-1 X m-2 BSA was reached.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Asma Inducida por Ejercicio/fisiopatología , Asma/fisiopatología , Pruebas de Provocación Bronquial/métodos , Hiperventilación/fisiopatología , Adolescente , Adulto , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Persona de Mediana EdadRESUMEN
A fluorogenic peptide, dansyl-D-Ala-Gly-Phe(pNO2)-Gly (DAGNPG), was synthesized as a selective substrate for the neutral metalloendopeptidase (EC 3.4.24.11) involved in enkephalin metabolism. This enzyme, designated "enkephalinase," cleaves the Gly-Phe(pNO2) peptide bond of DAGNPG (V = 0.65 mumol/mg protein/min and Km = 45 microM) leading to a fluorescence increase related to the disappearance of intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This change was used for quantitative measurements of "enkephalinase" activity in different tissues and determination of inhibitory potency of various compounds. The substrate is not cleaved by aminopeptidase or dipeptidylaminopeptidase activities and the assay itself is rapid, convenient, and sensitive.