RESUMEN
Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.
Asunto(s)
Carbapenémicos , Proteínas Hierro-Azufre , S-Adenosilmetionina , Vitamina B 12 , Carbapenémicos/biosíntesis , Carbapenémicos/química , Carbono , Proteínas Hierro-Azufre/química , Panteteína/química , S-Adenosilmetionina/química , Sulfuros , Vitamina B 12/químicaRESUMEN
Nature uses a diverse array of protein post-translational modifications (PTMs) to regulate protein structure, activity, localization, and function. Among them, protein 4'-phosphopantetheinylation derived from coenzyme A (CoA) is an essential PTM for the biosynthesis of fatty acids, polyketides, and nonribosomal peptides in prokaryotes and eukaryotes. To explore its functions, various chemical probes mimicking the natural structure of 4'-phosphopantetheinylation have been developed. In this minireview, we summarize these chemical probes and describe their applications in direct and metabolic labeling of proteins in bacterial and mammalian cells.
Asunto(s)
Coenzima A/química , Panteteína/análogos & derivados , Coenzima A/metabolismo , Modelos Moleculares , Estructura Molecular , Panteteína/química , Panteteína/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.
Asunto(s)
Proteína Transportadora de Acilo/química , Sintasas Poliquetidas/química , Ascomicetos/metabolismo , Dominio Catalítico , Reactivos de Enlaces Cruzados/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coli/metabolismo , Panteteína/química , Fosforilación , Propionatos/química , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Especificidad por SustratoRESUMEN
Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Coenzima A/farmacología , Difosfatos/farmacología , Diseño de Fármacos , Panteteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Aurora Quinasa A/metabolismo , Coenzima A/síntesis química , Coenzima A/química , Difosfatos/química , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Panteteína/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Dephospho coenzyme A (depCoA) is the last intermediate for CoA biosynthesis, and it can be used as a transcription initiator to prepare CoA-linked RNA by in vitro transcription. However, commercially available depCoA is expensive. We hereby describe a simple and efficient enzymatic synthesis of depCoA in a single-step from commercially available and inexpensive oxidized pantethine (Ox-Pan) and ATP. A plasmid (pCoaDAa) was constructed to co-express and co-purify two enzymes pantothenate kinase (PanK/coaA) and phosphopantetheine adenylyltransferase (PPAT/coaD). Starting from Ox-Pan and ATP, two different synthetic routes of one-pot reaction catalyzed by PanK and PPAT, followed by a simple column purification step, afforded depCoA and its oxidized dimer (Ox-depCoA) with high yields and purity. The simplicity and low cost of our method should make depCoA easily accessible to a broad scientific community, and promote research on CoA-related areas in biology and biomedicine.
Asunto(s)
Coenzima A/síntesis química , Nucleotidiltransferasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Secuencia de Bases , Técnicas de Química Sintética/métodos , Clonación Molecular/métodos , Escherichia coli/enzimología , Nucleotidiltransferasas/genética , Oxidación-Reducción , Panteteína/análogos & derivados , Panteteína/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Plásmidos/genéticaRESUMEN
Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.
Asunto(s)
Aciltransferasas/química , Aldehídos/química , Proteínas Bacterianas/química , Coenzima A/química , Inhibidores Enzimáticos/química , Panteteína/análogos & derivados , Polihidroxialcanoatos/química , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Aldehídos/síntesis química , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Biocatálisis , Biodegradación Ambiental , Coenzima A/síntesis química , Cupriavidus necator/química , Cupriavidus necator/enzimología , Perros , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Esterasas/química , Cinética , Lipasa/química , Simulación del Acoplamiento Molecular , Panteteína/síntesis química , Panteteína/química , Polihidroxialcanoatos/metabolismo , Homología Estructural de Proteína , Especificidad por Sustrato , Sulfolobus solfataricus/química , Sulfolobus solfataricus/enzimologíaRESUMEN
While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This KR naturally reduces both α- and ß-keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N-acetylcysteamine-bound ß-keto substrate to a D-ß-hydroxy product, but also an N-acetylcysteamine-bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a KR that naturally reduces a ß-keto group within a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase KRs reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase KRs, is predictive of the orientation of the resulting ß-hydroxyl group.
Asunto(s)
Aciltransferasas/química , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Sintasas Poliquetidas/química , Policétidos/química , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Panteteína/análogos & derivados , Panteteína/química , Péptido Sintasas , PolienosRESUMEN
We present a spherical micelle generated in a three-step sequence in which a farnesyl-pantetheine conjugate is phosphorylated, adenylated, and phosphorylated once more to generate a farnesyl-CoA amphiphile that self-assembles into spherical micelles. A sphere-to-fibril morphological switch is achieved by enzymatically transferring the farnesyl group of the farnesyl-CoA micelle onto a peptide via phosphopantetheinyl transferase to generate a peptide amphiphile. Each step in the sequence is followed with characterization by HPLC, MS, TEM, and DLS. This system offers an entry into cofactor-mediated peptide decoration by extending the principles of bioresponsive polymeric materials to sequential enzyme cascades.
Asunto(s)
Bacterias/enzimología , Nanoestructuras/química , Farnesol/química , Micelas , Estructura Molecular , Panteteína/químicaRESUMEN
Convenient two-step synthesis of conjugates of HS-CoA and D-pantetheine with aminooxy analogues of Spm, Spd and Put was suggested. The use of acetone linker provided target conjugates with quantitative yields. The activity of CoA-derived "bisubstrate" inhibitors being active at microM concentrations was at least 100 times better than that of corresponding derivatives of D-pantetheine.
Asunto(s)
Acetiltransferasas/síntesis química , Poliaminas/metabolismo , Espermidina/síntesis química , Espermina/síntesis química , Acetilcoenzima A/química , Acetiltransferasas/química , Bacterias/química , Bacterias/metabolismo , Bacterias/patogenicidad , Cinética , Panteteína/química , Poliaminas/química , Espermidina/química , Espermina/químicaRESUMEN
Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.
Asunto(s)
Colorantes Fluorescentes , Colorantes Fluorescentes/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/química , Fluorometría/métodos , Panteteína/análogos & derivados , Panteteína/metabolismo , Panteteína/químicaRESUMEN
Inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Staphylococcus aureus/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Animales , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Unión Competitiva , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Femenino , Pulmón/efectos de los fármacos , Pulmón/microbiología , Ratones , Modelos Moleculares , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Panteteína/análogos & derivados , Panteteína/química , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/microbiología , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/microbiología , Bibliotecas de Moléculas Pequeñas/química , Staphylococcus aureus/enzimología , Staphylococcus aureus/crecimiento & desarrollo , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/crecimiento & desarrollo , Muslo/microbiologíaRESUMEN
Acyl carrier proteins are critical components of fatty acid and polyketide biosynthesis. Their primary function is to shuttle intermediates between active sites via a covalently bound phosphopantetheine arm. Small molecules capable of acylating this prosthetic group will provide a simple and reversible means of introducing novel functionality onto carrier protein domains. A series of N-activated ß-lactams are prepared to examine site-specific acylation of the phosphopantetheine-thiol. In general, ß-lactams are found to be significantly more reactive than our previously studied ß-lactones. Selectivity for the holo over apo-form of acyl carrier proteins is demonstrated indicating that only the phosphopantetheine-thiol is modified. Incorporation of an N-propargyloxycarbonyl group provides an alkyne handle for conjugation to fluorophores and affinity labels. The utility of these groups for mechanistic interrogation of a critical step in polyketide biosynthesis is examined through comparison to traditional probes. In all, we expect the probes described in this study to serve as valuable and versatile tools for mechanistic interrogation.
Asunto(s)
Proteína Transportadora de Acilo/análisis , Colorantes Fluorescentes/química , beta-Lactamas/química , Acilación , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas , Panteteína/análogos & derivados , Panteteína/química , Compuestos de Sulfhidrilo/químicaRESUMEN
Protein-protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Escherichia coli/metabolismo , Resinas Sintéticas/química , Proteína Transportadora de Acilo/química , Espectrometría de Masas , Panteteína/química , Mapeo de Interacción de Proteínas , Shewanella/metabolismo , Especificidad por SustratoRESUMEN
In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4'-phosphopantetheine (4'-PP) to residue serine-36. We found that the long flexible 4'-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (kcat/Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.
Asunto(s)
Proteína Transportadora de Acilo , Panteteína , Cinética , Panteteína/química , Panteteína/metabolismo , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Escherichia coli/metabolismo , alfa-Amilasas/metabolismo , Proteínas Inmovilizadas/metabolismoRESUMEN
GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet_2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. Apo-GmACP3 exhibited slow precipitation and non-monomeric behavior by (15)N NMR relaxation measurements. Addition of 4'-phosphopantetheine (4'-PP) via enzymatic conversion by E. coli holo-ACP synthase resulted in stable >95% holo-GmACP3 that was characterized as monomeric by (15)N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4'-PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique characteristics, including an ordered 4'-PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conserved WDSLxH/N motif found in GmACP3 and its orthologs. The helix locations and the large hydrophobic cavity are more similar to medium- and long-chain acyl-ACPs than to other apo- and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggests that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially is involved in synthesis of secondary metabolites.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Geobacter/metabolismo , Lipopolisacáridos/biosíntesis , Panteteína/análogos & derivados , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Panteteína/química , Conformación ProteicaRESUMEN
ß-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a ß-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Escherichia coli/enzimología , Mycobacterium tuberculosis/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Coenzima A/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación , Palmitoil Coenzima A/metabolismo , Panteteína/análogos & derivados , Panteteína/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Especificidad por SustratoRESUMEN
Polyketide and nonribosomal peptides constitute important classes of small molecule natural products. Due to the proven biological activities of these compounds, novel methods for discovery and study of the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymes responsible for their production remains an area of intense interest, and proteomic approaches represent a relatively unexplored avenue. While these enzymes may be distinguished from the proteomic milieu by their use of the 4'-phosphopantetheine (PPant) post-translational modification, proteomic detection of PPant peptides is hindered by their low abundance and labile nature which leaves them unassigned using traditional database searching. Here we address key experimental and computational challenges to facilitate practical discovery of this important post-translational modification during shotgun proteomics analysis using low-resolution ion-trap mass spectrometers. Activity-based enrichment maximizes MS input of PKS/NRPS peptides, while targeted fragmentation detects putative PPant active sites. An improved data analysis pipeline allows experimental identification and validation of these PPant peptides directly from MS² data. Finally, a machine learning approach is developed to directly detect PPant peptides from only MS² fragmentation data. By providing new methods for analysis of an often cryptic post-translational modification, these methods represent a first step toward the study of natural product biosynthesis in proteomic settings.
Asunto(s)
Proteínas Bacterianas/química , Dominio Catalítico , Panteteína/análogos & derivados , Péptido Sintasas/química , Proteoma/química , Algoritmos , Inteligencia Artificial , Bacillus subtilis/química , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Panteteína/química , Panteteína/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptido Sintasas/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteómica , Curva ROC , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
L. major acyl carrier protein (ACP) is a mitochondrial protein, involved in fatty acid biosynthesis. The protein is expressed as an apo-protein, and post-translationally modified at Ser 37 by a 4'-Phosphopantetheinyl transferase. Crystal structure of the apo-form of the protein at pH 5.5 suggests a four helix bundle fold, typical of ACP's. However, upon lowering the pH to 5.0, it undergoes a conformational transition from α-helix to ß-sheet, and displays amyloid like properties. When left for a few days at room temperature at this pH, the protein forms fibrils, visible under Transmission electron microscopy (TEM). Using an approach combining NMR, biophysical techniques, and mutagenesis, we have identified a Phe residue present on helix II of ACP, liable for this change. Phosphopantetheinylation of LmACP, or mutation of Phe 45 to the corresponding residue in E. coli ACP (methionine), slows down the conformational change. Conversely, substitution of methionine 44 of E. coli ACP with a phenylalanine, causes enhanced ThT binding. Thus, we demonstrate the unique property of an exposed Phe in inducing, and phophopantetheine in inhibiting amyloidogenesis. Taken together, our study adds L. major acyl carrier protein to the list of ACPs that act as pH sensors.
Asunto(s)
Proteína Transportadora de Acilo/química , Leishmania major/química , Panteteína/análogos & derivados , Fenilalanina/química , Agregado de Proteínas , Proteínas Protozoarias/química , Panteteína/químicaRESUMEN
Acyltransferases are responsible for the selection and loading of acyl units onto carrier proteins in polyketide and fatty-acid biosynthesis. Despite the importance of protein-protein interactions between the acyltransferase and the carrier protein, structural information on acyltransferase-carrier protein interactions is limited because of the transient interactions between them. In the biosynthesis of the polyketide vicenistatin, the acyltransferase VinK recognizes the carrier protein VinL for the transfer of a dipeptidyl unit. The crystal structure of a VinK-VinL covalent complex formed with a 1,2-bismaleimidoethane cross-linking reagent has been determined previously. Here, the crystal structure of a VinK-VinL covalent complex formed with a pantetheine cross-linking probe is reported at 1.95â Å resolution. In the structure of the VinK-VinL-probe complex, the pantetheine probe that is attached to VinL is covalently connected to the side chain of the mutated Cys106 of VinK. The interaction interface between VinK and VinL is essentially the same in the two VinK-VinL complex structures, although the position of the pantetheine linker slightly differs. This structural observation suggests that interface interactions are not affected by the cross-linking strategy used.
Asunto(s)
Aciltransferasas/química , Aciltransferasas/metabolismo , Reactivos de Enlaces Cruzados/química , Panteteína/química , Panteteína/metabolismo , Dominios y Motivos de Interacción de Proteínas , Aciltransferasas/genética , Cristalografía por Rayos X , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Especificidad por SustratoRESUMEN
Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.