RESUMEN
Fatty acids are essential to most organisms and are made endogenously by the fatty acid synthase (FAS). FAS is an attractive target for antibiotics and many inhibitors are in clinical development. However, some gram-negative bacteria harbor an enzyme known as the acyl-acyl carrier protein synthetase (AasS), which allows them to scavenge fatty acids from the environment and shuttle them into FAS and ultimately lipids. The ability of AasS to recycle fatty acids may help pathogenic gram-negative bacteria circumvent FAS inhibition. We therefore set out to design and synthesize an inhibitor of AasS and test its effectiveness on an AasS enzyme from Vibrio harveyi, the most well studied AasS to date, and from Vibrio cholerae, a pathogenic model. The inhibitor C10-AMS [5'-O-(N-decanylsulfamoyl)adenosine], which mimics the tightly bound acyl-AMP reaction intermediate, was able to effectively inhibit AasS catalytic activity in vitro. Additionally, C10-AMS stopped the ability of Vibrio cholerae to recycle fatty acids from media and survive when its endogenous FAS was inhibited with cerulenin. C10-AMS can be used to study fatty acid recycling in other bacteria as more AasS enzymes continue to be annotated and provides a platform for potential antibiotic development.
Asunto(s)
Adenosina/síntesis química , Antibacterianos/síntesis química , Ligasas de Carbono-Azufre/metabolismo , Cólera/microbiología , Ácidos Grasos/metabolismo , Vibrio cholerae/fisiología , Vibrio/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Antibacterianos/farmacología , Catálisis , Cólera/tratamiento farmacológico , Desarrollo de Medicamentos , Ácido Graso Sintasas/metabolismo , Humanos , Especificidad por SustratoRESUMEN
Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anerobic, Gram-negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced the saturated fatty acids. The most abundant lipids were phosphatidylethanolamine (PE) and sulfonolipid (SL). Neither phosphatidylglycerol nor cardiolipin are present. PE synthesis is initiated by the PlsX/PlsY/PlsC pathway, whereas the SL pathway is related to sphingolipid biosynthesis. A. finegoldii incorporated medium-chain fatty acids (≤14 carbons) into PE and SL after their elongation, whereas long-chain fatty acids (≥16 carbons) were not elongated. Fatty acids >16 carbons were primarily incorporated into the 2-position of phosphatidylethanolamine at the PlsC step, the only biosynthetic enzyme that utilizes long-chain acyl-ACP. The ability to assimilate a broad-spectrum of fatty acid chain lengths present in the gut environment is due to the expression of two acyl-acyl carrier protein (ACP) synthetases. Acyl-ACP synthetase 1 had a substrate preference for medium-chain fatty acids and synthetase 2 had a substrate preference for long-chain fatty acids. This unique combination of synthetases allows A. finegoldii to utilize both the medium- and long-chain fatty acid nutrients available in the gut environment to assemble its membrane lipids.
Asunto(s)
Bacteroidetes/metabolismo , Ácidos Grasos/metabolismo , Microbioma Gastrointestinal , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Humanos , Lípidos/biosíntesis , Fosfatidiletanolaminas/biosíntesisRESUMEN
Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although ß-amino acid loading in the fluvirucinâ B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the ß-amino acid loading pathway in fluvirucinâ B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer ß-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of ß-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.
Asunto(s)
Aminoácidos/metabolismo , Desoxiazúcares/biosíntesis , Actinobacteria/química , Actinobacteria/enzimología , Aciltransferasas/química , Aciltransferasas/metabolismo , Aminoacilación , Ligasas de Carbono-Azufre/química , Ligasas de Carbono-Azufre/metabolismo , Carboxiliasas/química , Carboxiliasas/metabolismo , Catálisis , Lactamas , Estructura Molecular , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Dominios Proteicos , Especificidad por SustratoRESUMEN
Lysophospholipids (LPLs) are metabolic intermediates in bacterial phospholipid turnover. Distinct from their diacyl counterparts, these inverted cone-shaped molecules share physical characteristics of detergents, enabling modification of local membrane properties such as curvature. The functions of LPLs as cellular growth factors or potent lipid mediators have been extensively demonstrated in eukaryotic cells but are still undefined in bacteria. In the envelope of Gram-negative bacteria, LPLs are derived from multiple endogenous and exogenous sources. Although several flippases that move non-glycerophospholipids across the bacterial inner membrane were characterized, lysophospholipid transporter LplT appears to be the first example of a bacterial protein capable of facilitating rapid retrograde translocation of lyso forms of glycerophospholipids across the cytoplasmic membrane in Gram-negative bacteria. LplT transports lyso forms of the three bacterial membrane phospholipids with comparable efficiency, but excludes other lysolipid species. Once a LPL is flipped by LplT to the cytoplasmic side of the inner membrane, its diacyl form is effectively regenerated by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase (Aas). LplT-Aas also mediates a novel cardiolipin remodeling by converting its two lyso derivatives, diacyl or deacylated cardiolipin, to a triacyl form. This coupled remodeling system provides a unique bacterial membrane phospholipid repair mechanism. Strict selectivity of LplT for lyso lipids allows this system to fulfill efficient lipid repair in an environment containing mostly diacyl phospholipids. A rocker-switch model engaged by a pair of symmetric ion-locks may facilitate alternating substrate access to drive LPL flipping into bacterial cells. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.
Asunto(s)
Pared Celular/metabolismo , Bacterias Gramnegativas/metabolismo , Lipogénesis , Lisofosfolípidos/biosíntesis , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico , Ligasas de Carbono-Azufre/química , Ligasas de Carbono-Azufre/metabolismo , Lisofosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/química , Transducción de Señal , Especificidad por SustratoRESUMEN
Formaldehyde is a toxin and carcinogen that is both an environmental pollutant and an endogenous metabolite. Formaldehyde metabolism, which is probably essential for all aerobic cells, likely proceeds via multiple mechanisms, including via a glutathione-dependent pathway that is widely conserved in bacteria, plants and animals. However, it is unclear whether the first step in the glutathione-dependent pathway (i.e. formation of S-hydroxymethylglutathione (HMG)) is enzyme-catalysed. We report studies on glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans, which has been proposed to catalyse HMG formation from glutathione and formaldehyde on the basis of studies using NMR exchange spectroscopy (EXSY). Although we were able to replicate the EXSY results, time course experiments unexpectedly imply that GFA does not catalyse HMG formation under standard conditions. However, GFA was observed to bind glutathione using NMR and mass spectrometry. Overall, the results reveal that GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. Thus, it is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context.
Asunto(s)
Proteínas Bacterianas/química , Ligasas de Carbono-Azufre/química , Paracoccus denitrificans/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Datos de Secuencia MolecularRESUMEN
The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Chlamydia trachomatis/metabolismo , Ácidos Grasos/metabolismo , Fosfolípidos/biosíntesis , Proteínas Bacterianas/genética , Ligasas de Carbono-Azufre/genética , Chlamydia trachomatis/genética , Chlamydia trachomatis/fisiología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Cinética , Ácido Oléico/metabolismo , Ácido Palmítico/metabolismo , Especificidad por SustratoRESUMEN
Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.
Asunto(s)
Ligasas de Carbono-Azufre/metabolismo , Synechococcus/enzimología , Aclimatación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/genética , Ácidos Grasos no Esterificados/metabolismo , Luz , Lípidos de la Membrana/metabolismo , Mutación , Fotosíntesis/fisiología , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/fisiología , Complejo de Proteína del Fotosistema II/efectos de la radiación , Synechococcus/genética , Synechococcus/fisiología , Synechococcus/efectos de la radiaciónRESUMEN
Acyl carrier proteins (ACPs) are promiscuous small proteins that play essential roles in the biosynthesis of many natural products, but our understanding of how they interact with various enzymes within larger protein complexes is lacking. In this issue of Chemistry and Biology, Beld and coworkers describe an enzymatic labeling method that will allow tracking of ACPs as they interact with their partners both in vitro and vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Vibrio/enzimologíaRESUMEN
The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Vibrio/enzimología , Arabidopsis/enzimología , Proteínas Bacterianas/genética , Ligasas de Carbono-Azufre/genética , Coenzima A Ligasas/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Especificidad por Sustrato , Synechocystis/enzimología , Thermus thermophilus/enzimologíaRESUMEN
In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coliâ FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.
Asunto(s)
Coenzima A Ligasas/metabolismo , Ácidos Grasos/metabolismo , Xanthomonas campestris/enzimología , Acilcoenzima A/metabolismo , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Coenzima A Ligasas/genética , Coenzima A Ligasas/aislamiento & purificación , Difusión , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Prueba de Complementación Genética , Enzimas Multifuncionales/metabolismo , Familia de Multigenes , Mutación , Oxidación-Reducción , Enfermedades de las Plantas/microbiología , Transducción de Señal/genética , Especificidad por Sustrato , Tioléster Hidrolasas/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/crecimiento & desarrollo , Xanthomonas campestris/patogenicidadRESUMEN
Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their C-S bonds are formed by oxidative coupling reactions catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between histidine and cysteine (1 â 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 â 4 conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.
Asunto(s)
Ligasas de Carbono-Azufre/metabolismo , Ergotioneína/biosíntesis , Metilhistidinas/síntesis química , Betaína/análogos & derivados , Betaína/química , Catálisis , Cisteína/química , Ergotioneína/química , Ergotioneína/metabolismo , Histidina/análogos & derivados , Histidina/biosíntesis , Histidina/química , Histidina/metabolismo , Metilhistidinas/química , Metilhistidinas/metabolismo , Estructura Molecular , Oxidación-Reducción , Estereoisomerismo , Compuestos de Sulfhidrilo/químicaRESUMEN
Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ligasas de Carbono-Azufre/genética , Ácidos Grasos/metabolismo , Semillas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Cuphea/enzimología , Cuphea/genética , Ácidos Grasos/química , Mutación , Plantas Modificadas Genéticamente , Plastidios/enzimología , Plastidios/genética , Semillas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Triglicéridos/metabolismoRESUMEN
Free fatty acids are typically activated by thioesterification processes and catalyzed by the fatty acyl-CoA synthetase or fatty acyl-ACP synthetase. However, the routes for fatty acid activation in cyanobacteria are not well understood. In this investigation, the slr1609 gene, which encodes the fatty acid activation enzyme, was cloned from Synechocystis sp. PCC6803. This gene was identified by heterologous expression and in vitro enzymatic activity analyses. Different from previous reports stating that free fatty acids are only activated through the fatty acyl-ACP synthetases encoded by these genes in cyanobacteria, this gene was also proven to possess a fatty acyl-CoA synthetase activity, by in vitro enzymatic activity analyses and in vivo complementation experiments. The protein Slr1609 is located in both the cell membrane and the cytosol of Synechocystis sp. PCC6803. The differences in the transcriptional profiles between the wild type and the slr1609 deletion mutant strain were evaluated using microarray analyses. These analyses indicated that 299 differentially expressed genes are involved in fatty acid metabolism, photosynthesis, carbon fixation, stress tolerance and other metabolic processes. Our experiments demonstrate the observed compositional changes in the unsaturated fatty acids from the membrane lipids of the slr1609 deletion mutant when shifted from 30 to 24 °C.
Asunto(s)
Proteínas Bacterianas/metabolismo , Coenzima A Ligasas/metabolismo , Ácidos Grasos/metabolismo , Enzimas Multifuncionales/metabolismo , Synechocystis/enzimología , Synechocystis/genética , Proteínas Bacterianas/genética , Ligasas de Carbono-Azufre/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligasas/genética , Citosol/metabolismo , Ácidos Grasos/química , Ácidos Grasos Insaturados/metabolismo , Genes Bacterianos , Lípidos de la Membrana/química , Análisis por Micromatrices , Enzimas Multifuncionales/genética , Temperatura , Transcripción GenéticaRESUMEN
The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[(14)C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [(14)C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Synechocystis/enzimología , Ácido alfa-Linolénico/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Ligasas de Carbono-Azufre/genética , ADN Bacteriano/genética , Resistencia a Medicamentos , Transporte de Electrón , Activación Enzimática , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Transporte de Ácidos Grasos/genética , Recombinación Homóloga , Liposomas/metabolismo , Viabilidad Microbiana , Microscopía Electrónica de Rastreo , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/metabolismo , Fenotipo , Fotosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Synechocystis/efectos de los fármacos , Synechocystis/genética , Synechocystis/ultraestructura , Factores de Tiempo , Ácido alfa-Linolénico/farmacología , alfa-Tocoferol/metabolismoRESUMEN
Fatty acyl-AMP ligases (FAALs) activate fatty acids as acyladenylates, and subsequently catalyze their transfer onto the acyl carrier proteins (ACPs) of polyketide synthases (PKSs) or nonribosomal peptide synthetases to produce lipidic metabolites. Myxococcus xanthus contains a polyketide biosynthesis gene cluster in which putative FAAL (FtpD) and ACP (FtpC) genes are located close to a type III PKS (FtpA) gene. Here we describe the characterization of these three proteins in vitro. FtpD adenylated stearic acid and produced stearoyl-FtpC. The stearoyl moiety was then transferred to FtpA. When extender substrates (malonyl-CoA and methylmalonyl-CoA) were added to the reaction, the alkylresorcinol 5-heptadecyl-4-methyl-benzene-1,3-diol was synthesized. Further in vitro analysis indicated that FtpA produces an alkylresorcylic acid as the direct product, and that this decarboxylates to alkylresorcinol nonenzymatically. This is the first report of a FAAL supplying a long-chain fatty acyl-ACP starter substrate to a type III PKS.
Asunto(s)
Ligasas de Carbono-Azufre/metabolismo , Ácidos Grasos/biosíntesis , Myxococcus xanthus/enzimología , Sintasas Poliquetidas/metabolismo , Benceno/química , Benceno/metabolismo , Biocatálisis , Ligasas de Carbono-Azufre/genética , Biología Computacional , ADN Recombinante/genética , Familia de Multigenes , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Resorcinoles/metabolismo , Streptomyces lividans/genéticaRESUMEN
Ovothiols are histidine-derived thiols that were first isolated from marine invertebrates. We have identified a 5-histidylcysteine sulfoxide synthase (OvoA) as the first ovothiol biosynthetic enzyme and characterized OvoAs from Erwinia tasmaniensis and Trypanosoma cruzi . Homologous enzymes are encoded in more than 80 genomes ranging from proteobacteria to animalia.
Asunto(s)
Ligasas de Carbono-Azufre/metabolismo , Erwinia/enzimología , Metilhistidinas/metabolismo , Trypanosoma cruzi/enzimología , Animales , Ligasas de Carbono-Azufre/química , Ligasas de Carbono-Azufre/genética , Genoma/genética , Estructura Terciaria de ProteínaRESUMEN
Phthiocerol and phthiodiolone dimycocerosates (DIMs) and phenolic glycolipids (PGLs) are complex lipids located at the cell surface of Mycobacterium tuberculosis that play a key role in the pathogenicity of tuberculosis. Most of the genes involved in the biosynthesis of these compounds are clustered on a region of the M. tuberculosis chromosome, the so-called DIM + PGL locus. Among these genes, four ORFs encode FadD proteins, which activate and transfer biosynthetic intermediates onto various polyketide synthases that catalyze the formation of these lipids. In this study, we investigated the roles of FadD22, FadD26 and FadD29 in the biosynthesis of DIMs and related compounds. Biochemical characterization of the lipids produced by a spontaneous Mycobacterium bovis BCG mutant harboring a large deletion within fadD26 revealed that FadD26 is required for the production of DIMs but not of PGLs. Additionally, using allelic exchange recombination, we generated an unmarked M. tuberculosis mutant containing a deletion within fadD29. Biochemical analyses of this strain revealed that, like fadD22, this gene encodes a protein that is specifically involved in the biosynthesis of PGLs, indicating that both FadD22 and FadD29 are responsible for one particular reaction in the PGL biosynthetic pathway. These findings were also supported by in vitro enzymatic studies showing that these enzymes have different properties, FadD22 displaying a p-hydroxybenzoyl-AMP ligase activity, and FadD29 a fatty acyl-AMP ligase activity. Altogether, these data allowed us to precisely define the functions fulfilled by the various FadD proteins encoded by the DIM + PGL cluster.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas/metabolismo , Lípidos/biosíntesis , Mycobacterium tuberculosis/metabolismo , Ligasas de Carbono-Azufre/metabolismoRESUMEN
In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.
Asunto(s)
Ligasas de Carbono-Azufre/metabolismo , Ácidos Grasos/metabolismo , Synechococcus/enzimología , Synechocystis/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Azufre/genética , Clonación Molecular , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , ADN Bacteriano/genética , Técnicas de Inactivación de Genes , Filogenia , Alineación de Secuencia , Especificidad por Sustrato , Synechococcus/genética , Synechocystis/genéticaRESUMEN
Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.
Asunto(s)
Aciltransferasas/metabolismo , Ligasas de Carbono-Azufre/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos/biosíntesis , Lípido A/biosíntesis , Vibrio/enzimología , Aciltransferasas/genética , Ligasas de Carbono-Azufre/genética , Escherichia coli/enzimología , Especificidad por Sustrato , Vibrio/metabolismoRESUMEN
Daptomycin and A21987C antibiotics are branched, cyclic, nonribosomally assembled acidic lipodepsipeptides produced by Streptomyces roseosporus. The antibacterial activity of daptomycin against gram-positive bacteria strongly depends on the nature of the N-terminal fatty acid moiety. Two genes, dptE and dptF, localized upstream of the daptomycin nonribosomal peptide synthetase genes, are thought to be involved in the lipidation of daptomycin. Here we describe the cloning, heterologous expression, purification and biochemical characterization of the enzymes encoded by these genes. DptE was proven to preferentially activate branched mid- to long-chain fatty acids under ATP consumption, and these fatty acids are subsequently transferred onto DptF, the cognate acyl carrier protein. Additionally, we demonstrate that lipidation of DptF by DptE in trans is based on specific protein-protein interactions, as DptF is favored over other acyl carrier proteins. Study of DptE and DptF may provide useful insights into the lipidation mechanism, and these enzymes may be used to generate novel daptomycin derivatives with altered fatty acids.