RESUMEN
Las anomalías vasculares de la órbita (AVO) son un grupo heterogéneo de patologías que pueden presentarse con frecuencia en el cono orbitario, la región periorbitaria o dentro de la órbita misma. Las AVO se dividen en tumores y malformaciones. Su presentación clínica más frecuente es el exoftalmos, asociado o no a alteración del eje visual. Además, pueden presentar complicaciones agudas, como hemorragia intralesional o celulitis entre las más frecuentes, y complicaciones crónicas, como ambliopía y afectación de la agudeza visual a largo plazo. La evolución de las técnicas de imágenes, el uso de nuevos fármacos y la utilización de innovadores procedimientos en radiología intervencionista han posibilitado obtener una mejora significativa en los procesos diagnósticos y terapéuticos de estos pacientes, permitiendo un diagnóstico y tratamiento preciso.
Orbital vascular anomalies (OVAs) are a heterogeneous group of disorders frequently found in the orbital cone, the periorbital region, or within the orbit itself. OVAs are divided into tumors and malformations. The most frequent clinical presentation is exophthalmos, associated or not with an alteration of the visual axis. They may also cause acute complications, being intralesional bleeding or cellulitis the most frequent, and chronic complications, such as amblyopia and long-term visual acuity impairment. The development of imaging techniques, the use of new drugs, and the implementation of innovative procedures in interventional radiology have resulted in a significant improvement in the diagnostic and therapeutic approaches to these patients, essential to an accurate diagnosis and management.
Asunto(s)
Humanos , Niño , Exoftalmia , Malformaciones Vasculares/terapia , Malformaciones Vasculares/diagnóstico por imagen , Órbita/irrigación sanguínea , Órbita/patología , Agudeza Visual , Hemorragia/patologíaRESUMEN
Orbital vascular anomalies (OVAs) are a heterogeneous group of disorders frequently found in the orbital cone, the periorbital region, or within the orbit itself. OVAs are divided into tumors and malformations. The most frequent clinical presentation is exophthalmos, associated or not with an alteration of the visual axis. They may also cause acute complications, being intralesional bleeding or cellulitis the most frequent, and chronic complications, such as amblyopia and long-term visual acuity impairment. The development of imaging techniques, the use of new drugs, and the implementation of innovative procedures in interventional radiology have resulted in a significant improvement in the diagnostic and therapeutic approaches to these patients, essential to an accurate diagnosis and management.
Las anomalías vasculares de la órbita (AVO) son un grupo heterogéneo de patologías que pueden presentarse con frecuencia en el cono orbitario, la región periorbitaria o dentro de la órbita misma. Las AVO se dividen en tumores y malformaciones. Su presentación clínica más frecuente es el exoftalmos, asociado o no a alteración del eje visual. Además, pueden presentar complicaciones agudas, como hemorragia intralesional o celulitis entre las más frecuentes, y complicaciones crónicas, como ambliopía y afectación de la agudeza visual a largo plazo. La evolución de las técnicas de imágenes, el uso de nuevos fármacos y la utilización de innovadores procedimientos en radiología intervencionista han posibilitado obtener una mejora significativa en los procesos diagnósticos y terapéuticos de estos pacientes, permitiendo un diagnóstico y tratamiento preciso.
Asunto(s)
Exoftalmia , Malformaciones Vasculares , Humanos , Niño , Malformaciones Vasculares/diagnóstico por imagen , Malformaciones Vasculares/terapia , Órbita/irrigación sanguínea , Órbita/patología , Agudeza Visual , Hemorragia/patologíaRESUMEN
Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as "lipoyl-relay", requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.
Asunto(s)
Redes y Vías Metabólicas , Ácido Tióctico , Trypanosoma brucei brucei , Bacillus subtilis/metabolismo , Ligasas/metabolismo , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/metabolismo , Ácido Tióctico/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below â¼30°C. Membrane fluidity signals are transmitted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of membrane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased mutagenic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every transmembrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the metastable 2-HCC domain of the DesK signaling state.IMPORTANCE Signal sensing and transduction is an essential biological process for cell adaptation and survival. Histidine kinases (HK) are the sensory proteins of two-component systems that control many bacterial responses to different stimuli, like environmental changes. Here, we focused on the HK DesK from Bacillus subtilis, a paradigmatic example of a transmembrane thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C. DesK provides a tractable system for investigating the mechanism of transmembrane signaling, one of the majors interrogates in biology to date. Our studies demonstrate that transmembrane proline residues modulate the conformational switch of a 2-helix coiled-coil (2-HCC) structural motif that controls input-output in a variety of HK. Our results highlight the relevance of proline residues within sensor domains and could inspire investigations of their role in different signaling proteins.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Histidina Quinasa/química , Histidina Quinasa/metabolismo , Prolina/metabolismo , Secuencias de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/química , Membrana Celular/genética , Histidina Quinasa/genética , Fluidez de la Membrana , Dominios ProteicosRESUMEN
Two component systems, composed of a receptor histidine kinase and a cytoplasmic response regulator, regulate pivotal cellular processes in microorganisms. Here we describe a new screening procedure for the identification of amino acids that are crucial for the functioning of DesK, a prototypic thermosensor histidine kinase from Bacillus subtilis. This experimental strategy involves random mutagenesis of the membrane sensor domain of the DesK coding sequence, followed by the use of a detection procedure based on changes in the colony morphogenesis that take place during the sporulation programme of B. subtilis. This method permitted us the recovery of mutants defective in DesK temperature sensing. This screening approach could be applied to all histidine kinases of B. subtilis and also to kinases of other bacteria that are functionally expressed in this organism. Moreover, this reporter assay could be expanded to develop reporter assays for a variety of transcriptionally regulated systems.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacillus subtilis/genética , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Mutagénesis , Mutación , TemperaturaRESUMEN
Bacillus subtilis responds to a sudden decrease in temperature by transiently inducing the expression of the des gene encoding for a lipid desaturase, Δ5-Des, which introduces a double bond into the acyl chain of preexisting membrane phospholipids. This Δ5-Des-mediated membrane remodeling is controlled by the cold-sensor DesK. After cooling, DesK activates the response regulator DesR, which induces transcription of des. We show that inhibition of fatty acid synthesis by the addition of cerulenin, a potent and specific inhibitor of the type II fatty acid synthase, results in increased levels of short-chain fatty acids (FA) in membrane phospholipids that lead to inhibition of the transmembrane-input thermal control of DesK. Furthermore, reduction of phospholipid synthesis by conditional inactivation of the PlsC acyltransferase causes significantly elevated incorporation of long-chain FA and constitutive upregulation of the des gene. Thus, we provide in vivo evidence that the thickness of the hydrophobic core of the lipid bilayer serves as one of the stimulus sensed by the membrane spanning region of DesK.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Cerulenina/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Proteínas de la Membrana/metabolismo , Bacillus subtilis/efectos de la radiación , Membrana Celular/metabolismo , Frío , Ácido Graso Desaturasas/metabolismo , Transducción de SeñalRESUMEN
In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/genética , Vías Biosintéticas/genética , Genes Bacterianos , Ácido Tióctico/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM and ywfL, which we have renamed lplJ, lipM and lipL respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to Escherichia coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulphur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilisΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coliΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion article provide evidence that LipM and LipL catalyse sequential reactions in a novel pathway for lipoic acid biosynthesis.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/genética , Vías Biosintéticas/genética , Genes Bacterianos , Ácido Tióctico/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética , Modelos BiológicosRESUMEN
Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/fisiología , Frío , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Membrana Celular , Histidina Quinasa , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Proteínas Quinasas/química , Proteínas Quinasas/fisiologíaRESUMEN
Bacillus subtilis, which grows under aerobic conditions, employs fatty acid desaturase (Des) to fluidize its membrane when subjected to temperature downshift. Des requires molecular oxygen for its activity, and its expression is regulated by DesK-DesR, a two-component system. Transcription of des is induced by the temperature downshift and is decreased when membrane fluidity is restored. B. subtilis is also capable of anaerobic growth by nitrate or nitrite respiration. We studied the mechanism of cold adaptation in B. subtilis under anaerobic conditions that were predicted to inhibit Des activity. We found that in anaerobiosis, in contrast to aerobic growth, the induction of des expression after temperature downshift (from 37 degrees C to 25 degrees C) was not downregulated. However, the transfer from anaerobic to aerobic conditions rapidly restored the downregulation. Under both aerobic and anaerobic conditions, the induction of des expression was substantially reduced by the addition of external fluidizing oleic acid and was fully dependent on the DesK-DesR two-component regulatory system. Fatty acid analysis proved that there was no desaturation after des induction under anaerobic conditions despite the presence of high levels of the des protein product, which was shown by immunoblot analysis. The cold adaptation of B. subtilis in anaerobiosis is therefore mediated exclusively by the increased anteiso/iso ratio of branched-chain fatty acids and not by the temporarily increased level of unsaturated fatty acids that is typical under aerobic conditions. The degrees of membrane fluidization, as measured by diphenylhexatriene fluorescence anisotropy, were found to be similar under both aerobic and anaerobic conditions.
Asunto(s)
Adaptación Fisiológica , Bacillus subtilis/fisiología , Frío , Regulación Bacteriana de la Expresión Génica , Aerobiosis , Anaerobiosis , Bacillus subtilis/metabolismo , Proteínas Bacterianas/biosíntesis , Membrana Celular/química , Ácido Graso Desaturasas/biosíntesis , Ácidos Grasos/metabolismo , Fluidez de la Membrana , Transducción de SeñalAsunto(s)
Neoplasias de la Retina/diagnóstico , Retinoblastoma/diagnóstico , Femenino , Humanos , LactanteRESUMEN
Niña de 7 meses, previamente sana, traída a la consulta por leucocoria de 2 meses de evolución.La paciente no presenta dolor ocular ni síntomas concomitantes. Al examen oftalmológico se constata ausencia de fijación y esotropia del ojo derechocon ausencia de reflejo rojo.
Asunto(s)
Humanos , Femenino , Lactante , Diagnóstico Diferencial , Exotropía , Retinoblastoma/diagnóstico , Retinoblastoma/terapiaRESUMEN
Lipoic acid is an essential cofactor required for the function of key metabolic pathways in most organisms. We report the characterization of a Bacillus subtilis mutant obtained by disruption of the lipA (yutB) gene, which encodes lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor. The function of lipA was inferred from the results of genetic and physiological experiments, and this study investigated its role in B. subtilis fatty acid metabolism. Interrupting lipoate-dependent reactions strongly inhibits growth in minimal medium, impairing the generation of branched-chain fatty acids and leading to accumulation of copious amounts of straight-chain saturated fatty acids in B. subtilis membranes. Although depletion of LipA induces the expression of the Delta5 desaturase, controlled by a two-component system that senses changes in membrane properties, the synthesis of unsaturated fatty acids is insufficient to support growth in the absence of precursors for branched-chain fatty acids. However, unsaturated fatty acids generated by deregulated overexpression of the Delta5 desaturase functionally replaces lipoic acid-dependent synthesis of branched-chain fatty acids. Furthermore, we show that the cold-sensitive phenotype of a B. subtilis strain deficient in Delta5 desaturase is suppressed by isoleucine only if LipA is present.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ácidos Grasos/biosíntesis , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Vías Biosintéticas , Medios de Cultivo/química , Eliminación de Gen , Técnicas de Inactivación de GenesRESUMEN
Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Quinasas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Catálisis , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Histidina Quinasa , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , TemperaturaRESUMEN
Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. We are investigating how Bacillus subtilis cells sense and transduce low-temperature signals to adjust its gene expression. One important step has been accomplished in the dissection of a novel pathway for the adjustment of unsaturated fatty acid synthesis in B.subtilis, termed the Des pathway. It responds to a decrease in growth temperature by enhancing the expression of the des gene, coding for an acyl-lipid desaturase. The Des pathway is uniquely and stringently regulated by a tw-component system composed of a membrane-associated kinase, DesK, and a soluble transcriptional activator, DesR. The temperature sensing ability of the DesK protein is regulated by the extent of disorder within the membrane lipid bilayer. In this work, we present the mechanism by which the sensor protein DesK controls the signal decay of its cognate partner, DesR, and how this response regulator activates transcription of its target promoter. The results of these analysis will be presented and discussed in the context of transcriptional regulation of membrane fluidity homeostasis.
Asunto(s)
Bacillus subtilis/fisiología , Frío , Fluidez de la Membrana/fisiología , Transducción de Señal/fisiologíaRESUMEN
Most fatty acid desaturases are members of a large superfamily of integral membrane, O2-dependent, iron-containing enzymes that insert double bonds into previously synthesized fatty acyl chains. The cold shock-induced, membrane-bound desaturase from Bacillus subtilis (Delta5-Des) uses existing phospholipids as substrates to introduce a cis-double bond at the fifth position of the fatty acyl chain. While essentially no three-dimensional structural information is available for these difficult-to-purify enzymes, experimental analysis of the topology of Delta5-Des has provided a model that might be extended to most acyl-lipid desaturases. In addition, studies of the cold-induced expression of Delta5-Des led to the identification of a two-component system composed of a membrane-associated kinase, DesK, and a transcriptional regulator, DesR, which stringently controls the transcription of the des gene, coding for the desaturase. A model for sensing and transduction of low-temperature signals has emerged from our results, which we discuss in the context of transcriptional regulation of membrane lipid fluidity homeostasis.
Asunto(s)
Bacillus subtilis/enzimología , Ácido Graso Desaturasas/metabolismo , Fosfolípidos/metabolismo , Adaptación Fisiológica , TemperaturaAsunto(s)
Bacillus subtilis/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Fluidez de la Membrana/fisiología , Lípidos de la Membrana/química , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Escherichia coli/fisiología , Ácidos Grasos/metabolismo , Respuesta al Choque Térmico , TemperaturaRESUMEN
CysH1 from Bacillus subtilis encodes a 3'-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa. Recombinant B. subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants. The enzyme has a higher affinity for PAPS (K(m)PAPS 6.4 microm Trx-saturating, 10.7 microm Grx-saturating) than for APS (K(m) APS 28.7 microm Trx-saturating, 105 microm Grx-saturating) at a V(max) ranging from 280 to 780 nmol sulfite mg(-1) min(-1). The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (K(cat)/K(m) 2.7 x 10(4)-3.6 x 10(4) liter mol(-1) s(-1). B. subtilis SNR contains one 4Fe-4S cluster per polypeptide chain. SNR activity and color were lost rapidly upon exposure to air or upon dilution. Mössbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster. Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS. Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS. The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO(3) formation under aerobiosis.
Asunto(s)
Bacillus subtilis/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Proteínas Hierro-Azufre/química , Oxidación-Reducción , Oxidorreductasas/genética , Oxígeno/farmacología , Proteínas Recombinantes , Espectrofotometría , Espectroscopía de Mossbauer , Especificidad por SustratoRESUMEN
The Des pathway of Bacillus subtilis regulates the expression of the acyl-lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids from saturated phospholipid precursors. Activation of this pathway takes place when cells are shifted to low growth temperature or when they are grown in minimal media in the absence of isoleucine supplies. The master switch for the Des pathway is a two-component regulatory system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. We propose that both, a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism, involving the ability of DesK to sense a decrease in membrane fluidity.
Asunto(s)
Bacillus subtilis/metabolismo , Ácidos Grasos/metabolismo , delta-5 Desaturasa de Ácido Graso , Ácido Graso Desaturasas/metabolismo , Lípidos , Modelos Biológicos , Temperatura , Transcripción GenéticaRESUMEN
The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA). The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.