RESUMEN
The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C4-C10) or acyl-ACPs (C6-C10). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from ß-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Acil-Butirolactonas , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Ácidos Grasos/metabolismo , Bacterias/metabolismo , Escherichia coli/metabolismo , Acetilcoenzima A/metabolismoRESUMEN
Acinetobacter baumannii is one of the most serious opportunistic pathogens according to WHO. The difference between bacterial and mammalian fatty acid biosynthesis pathways makes FASII enzymes attractive targets in drug discovery. 3-oxoacyl-[acyl-carrier-protein] synthase I (FabB) from the FAS II pathway catalyze the condensation of malonyl ACP with acyl-ACP, and elongates the fatty acid chain by two carbons. To investigate potential inhibitors of the A. baumannii FabB, we used computational approaches including homology modeling, high-throughput virtual screening, molecular docking, molecular dynamics simulations, and MM-GBSA free energy calculations. After the high-throughput virtual screening, the resulting ligands were further screened using the QM-polarized ligand docking (QPLD) and induced fit docking (IFD) approaches. Molecular dynamics simulations were performed for 100 ns. And according to binding free energy calculations, we have identified nine compounds with the best binding affinities. Three of these compounds were selected for an additional 1 µs MD simulation to assess ligand stability. Two of them named L6 and L7 showed promised stability and affinity to the target. Here, we present novel compounds against A. baumannii FabB via structure-based computational approaches. These compounds might pave the way for the design of new lead structures and inhibitors for multidrug-resistant A. baumannii.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Acinetobacter baumannii , Simulación del Acoplamiento Molecular , Proteína Transportadora de Acilo , Glucógeno Sintasa , Ligandos , Simulación de Dinámica Molecular , Ácidos Grasos , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/químicaRESUMEN
Exploring new herbicide targets based on natural product derivatives is an important research aspect for the generation of innovative pesticides. Ferulic acid ethyl ester (FAEE), a natural product derivative from ferulic acid, has significant herbicidal activity mainly by inhibiting the normal growth of weed seedling roots. However, the FAEE target protein underlying its herbicidal activity has not been identified. In this study, we synthesized an FAEE probe to locate its site of action. We discovered that FAEE entry point was via the root tips. Fourteen major binding proteins were identified using Drug affinity responsive target stability (DARTS) combined with LC-MS/MS, which included 3-ketoacyl-acyl carrier protein synthase I (KAS I) and phenylalanine ammonia-lyase I (PAL I). The KAS I and PAL I proteins/genes expression was changed significantly after exposure to FAEE, as evidenced by combined transcriptomic and proteomic analysis. A molecular docking assay indicated that KAS I and FAEE had a strong binding ability. Combined with previous studies on FAEE mechanism of action, and based on our results, we conclude that FAEE targeting KAS I lead to the blockage of the fatty acid synthesis pathway and result in plant death.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Herbicidas , Raíces de Plantas , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Cromatografía Liquida , Ésteres , Ácidos Grasos/metabolismo , Herbicidas/farmacología , Simulación del Acoplamiento Molecular , Proteómica , Espectrometría de Masas en Tándem , Raíces de Plantas/efectos de los fármacosRESUMEN
Recent studies have reported the ß-ketoacyl-acyl carrier protein KasA as a druggable target for Mycobacterium tuberculosis. This review summarizes the current status of major classes of KasA inhibitors with an emphasis on significant contributions from structure-based design methods leveraging X-ray crystal structures of KasA alone and in complex with inhibitors. The issues addressed within each inhibitor class are discussed while detailing the characterized interactions with KasA and structure-activity relationships. A critical analysis of these findings should lay the foundation for new KasA inhibitors to study the basic biology of M. tuberculosis and to form the basis of new antitubercular molecules of clinical significance with activity against drug-sensitive and drug-resistant infections.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Mycobacterium tuberculosis , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Proteína Transportadora de Acilo , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Proteínas de Escherichia coli , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Carbono/metabolismo , Catálisis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II , Ácidos Grasos Insaturados/metabolismoRESUMEN
Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with ß-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca2+ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α1α2 loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α2 and α3-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α2-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein-protein interactions.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/metabolismo , Proteína Transportadora de Acilo/química , Antibacterianos/química , Sitios de Unión , Calcio/química , Dicroismo Circular , Farmacorresistencia Microbiana , Ácidos Grasos/química , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Metales/química , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Electricidad EstáticaRESUMEN
Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB ( PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E.coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Antibacterianos/farmacología , Escherichia coli , Ácidos Grasos , Pseudomonas aeruginosaRESUMEN
BACKGROUND: Antimicrobial resistance is a persistent problem regarding infection treatment and calls for developing new antimicrobial agents. Inhibition of bacterial ß-ketoacyl acyl carrier protein synthase III (FabH), which catalyzes the condensation reaction between a CoAattached acetyl group and an ACP-attached malonyl group in bacteria is an interesting strategy to find new antibacterial agents. OBJECTIVE: The aim of this work was to design and synthesize arylsulfonylhydrazones potentially FabH inhibitors and evaluate their antimicrobial activity. METHODS: MIC50 values of sulfonylhydrazones against E. coli and S. aureus were determined. Antioxidant activity was evaluated by DPPH (1-1'-diphenyl-2-picrylhydrazyl) assay and cytotoxicity against LL24 lung fibroblast cells was verified by MTT method. Principal component analysis (PCA) was performed in order to suggest a structure-activity relationship. Molecular docking allowed to propose sulfonylhydrazones interactions with FabH. RESULTS: The most active compound showed activity against S. aureus and E. coli, with MIC50 = 0.21 and 0.44 µM, respectively. PCA studies correlated better activity to lipophilicity and molecular docking indicated that sulfonylhydrazone moiety is important to hydrogen-bond with FabH while methylcatechol ring performs π-π stacking interaction. The DPPH assay revealed that some sulfonylhydrazones derived from the methylcatechol series had antioxidant activity. None of the evaluated compounds was cytotoxic to human lung fibroblast cells, suggesting that the compounds might be considered safe at the tested concentration. CONCLUSION: Arylsufonylhydrazones is a promising scaffold to be explored for the design of new antimicrobial agents.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Hidrazonas/farmacología , Sulfonamidas/farmacología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/química , Acetiltransferasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Dominio Catalítico , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/metabolismo , Hidrazonas/síntesis química , Hidrazonas/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Análisis de Componente Principal , Unión Proteica , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismoRESUMEN
Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, ß-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Acetiltransferasas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Acido Graso Sintasa Tipo II/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/aislamiento & purificación , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Catálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis , Mutación , Conformación Proteica , Proteínas RecombinantesRESUMEN
The bacterial fatty acid pathway is essential for membrane synthesis and a range of other metabolic and cellular functions. The ß-ketoacyl-ACP synthases carry out the initial elongation reaction of this pathway, utilizing acetyl-CoA as a primer to elongate malonyl-ACP by two carbons, and subsequent elongation of the fatty acyl-ACP substrate by two carbons. Here we describe the structures of the ß-ketoacyl-ACP synthase I from Brucella melitensis in complex with platencin, 7-hydroxycoumarin, and (5-thiophen-2-ylisoxazol-3-yl)methanol. The enzyme is a dimer and based on structural and sequence conservation, harbors the same active site configuration as other ß-ketoacyl-ACP synthases. The platencin binding site overlaps with the fatty acyl compound supplied by ACP, while 7-hydroxyl-coumarin and (5-thiophen-2-ylisoxazol-3-yl)methanol bind at the secondary fatty acyl binding site. These high-resolution structures, ranging between 1.25 and 1.70 å resolution, provide a basis for in silico inhibitor screening and optimization, and can aid in rational drug design by revealing the high-resolution binding interfaces of molecules at the malonyl-ACP and acyl-ACP active sites.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Aminofenoles/farmacología , Brucella melitensis/enzimología , Inhibidores Enzimáticos/farmacología , Compuestos Policíclicos/farmacología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Secuencia de Aminoácidos , Aminofenoles/química , Brucella melitensis/química , Brucella melitensis/metabolismo , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Compuestos Policíclicos/química , Conformación Proteica/efectos de los fármacos , Especificidad por SustratoRESUMEN
The discovery of new antimicrobial agents is extremely needed to overcome multidrug-resistant bacterial and tuberculosis infections. In the present study, eight novel substituted urea derivatives (10a-10h) containing disulfide bond were designed, synthesized and screened for their inâ vitro antimicrobial activities on standard strains of Gram-positive and Gram-negative bacteria as well as on Mycobacterium tuberculosis. According to the obtained results, antibacterial effects of the compounds were found to be considerably better than their antimycobacterial activities along with their weak cytotoxic effects. Molecular docking studies were performed to gain insights into the antibacterial activity mechanism of the synthesized compounds. The interactions and the orientation of compound 10a (1,1'-((disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-phenylurea)) were found to be highly similar to the original ligand within the binding pocket E. faecalis ß-ketoacyl acyl carrier protein synthase III (FabH). Finally, a theoretical study was established to predict the physicochemical properties of the compounds.
Asunto(s)
Antibacterianos/síntesis química , Disulfuros/química , Urea/análogos & derivados , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Animales , Antibacterianos/farmacología , Antituberculosos/síntesis química , Antituberculosos/farmacología , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Enterococcus faecalis/enzimología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Células RAW 264.7 , Relación Estructura-Actividad , Urea/farmacologíaRESUMEN
The enzyme ß-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115-147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Proteínas Bacterianas/química , Isoenzimas/química , Proteínas Mutantes/química , Mutación , Mycobacterium tuberculosis/enzimología , Tuberculosis/microbiología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Proteínas Bacterianas/genética , Isoenzimas/genética , Simulación de Dinámica Molecular , Proteínas Mutantes/genética , Conformación Proteica , Tuberculosis/genética , Tuberculosis/metabolismoRESUMEN
Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and ß-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Proteína Transportadora de Acilo/metabolismo , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Proteína Transportadora de Acilo/química , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Acido Graso Sintasa Tipo II/química , Modelos Moleculares , Unión ProteicaRESUMEN
Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against Propionibacterium acnes-induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of P. acnes and P. acnes-induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes. Secreted embryonic alkaline phosphatase assay confirmed that the anti-inflammatory activity of phloretin is associated with the P. acnes-stimulated TLR2-mediated NF-κB signaling pathway. Phloretin significantly decreased the level of phosphorylated c-Jun N-terminal kinase (JNK), showing a binding affinity of 1.184 × 10-5 M-1. We also found that phloretin binds with micromolar affinity to P. acnes ß-ketoacyl acyl carrier protein (ACP) synthase III (KAS III), an enzyme involved in fatty acid synthesis. Conformation-sensitive native polyacrylamide gel electrophoresis showed that phloretin reduced KAS III-mediated 3-ketoacyl ACP production by over 66%. A docking study revealed that phloretin interacts with the active sites of JNK1 and KAS III, suggesting their involvement in P. acnes-induced inflammation and their potential as targets for the antibacterial activity of phloretin. These results demonstrate that phloretin may be useful in the prevention or treatment of P. acnes infection.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Bacterias Grampositivas/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Floretina/farmacología , Propionibacterium acnes/efectos de los fármacos , Enfermedades Cutáneas Bacterianas/metabolismo , Enfermedades Cutáneas Bacterianas/microbiología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Antibacterianos/química , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Floretina/química , Propionibacterium acnes/enzimología , Propionibacterium acnes/inmunología , Unión Proteica , Enfermedades Cutáneas Bacterianas/tratamiento farmacológico , Relación Estructura-Actividad , Receptor Toll-Like 2/metabolismoRESUMEN
ß-Ketoacyl-acyl carrier protein synthase-III (KAS-III) and its homologues are thiolase-fold proteins that typically behave as homodimers functioning in diverse thioester-based reactions for C-C, C-O, or C-N bond formation. Here, we report an exception observed in the biosynthesis of lipstatin. During the establishment of the C22 aliphatic skeleton of this ß-lactone lipase inhibitor, LstA and LstB, which both are KAS-III homologues but phylogenetically distinct from each other, function together by forming an unusual heterodimer to catalyze a nondecarboxylating Claisen condensation of C8 and C14 fatty acyl-CoA substrates. The resulting C22 α-alkyl ß-ketoacid, which is unstable and tends to be spontaneously decarboxylated to a shunt C21 hydrocarbon product, is transformed by the stereoselective ß-ketoreductase LstD into a relatively stable C22 α-alkyl ß-hydroxyacid for further transformation. LstAB activity tolerates changes in the stereochemistry, saturation degree, and thioester form of both long-chain fatty acyl-CoA substrates. This flexibility, along with the characterization of catalytic residues, benefits our investigations into the individual roles of the two KAS-III homologues in the heterodimer-catalyzed reactions. The large subunit LstA contains a characteristic Cys-His-Asn triad and likely reacts with C8 acyl-CoA to form an acyl-Cys enzyme intermediate. In contrast, the small subunit LstB lacks this triad but possesses a catalytic Glu residue, which can act on the C8 acyl-Cys enzyme intermediate in a substrate-dependent manner, either as a base for Cα deprotonation or as a nucleophile for a Michael-type addition-initiated cascade reaction, to produce an enolate anion for head-to-head assembly with C14 acyl-CoA through a unidirectional nucleophilic substitution. Uncovering LstAB catalysis draws attention to thiolase-fold proteins that are noncanonical in both active form and catalytic reaction/mechanism. LstAB homologues are widespread in bacteria and remain to be functionally assigned, generating great interest in their corresponding products and associated biological functions.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acilcoenzima A/metabolismo , Lactonas/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Acilcoenzima A/química , Humanos , Lactonas/química , Estructura Molecular , Especificidad por SustratoRESUMEN
Propionibacterium acnes is an anaerobic gram-positive bacterium found in the niche of the sebaceous glands in the human skin, and is a causal pathogen of inflammatory skin diseases as well as periprosthetic joint infection. To gain effective control of P. acnes, a deeper understanding of the cellular metabolism mechanism involved in its ability to reside in this unique environment is needed. P. acnes exhibits typical cell membrane features of gram-positive bacteria, such as control of membrane fluidity by branched-chain fatty acids (BCFAs). Branching at the iso- or anteiso-position is achieved by incorporation of isobutyryl- or 2-methyl-butyryl-CoA via ß-ketoacyl acyl carrier protein synthase (KAS III) from fatty acid synthesis. Here, we determined the crystal structure of P. acnes KAS III (PaKAS III) at the resolution of 1.9â¯Å for the first time. Conformation-sensitive urea polyacrylamide gel electrophoresis and tryptophan fluorescence quenching experiments confirmed that PaKAS III prefers isobutyryl-CoA as the acetyl-CoA, and the unique shape of the active site cavity complies with incorporation of branched-short chain CoAs. The determined structure clearly illustrates how BCFA synthesis is achieved in P. acnes. Moreover, the unique shape of the cavity required for the branched-chain primer can be invaluable in designing novel inhibitors of PaKAS III and developing new specifically targeted antibiotics.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos Grasos/metabolismo , Propionibacterium acnes/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Vías Biosintéticas , Cristalografía por Rayos X , Ácidos Grasos/química , Modelos Moleculares , Propionibacterium acnes/química , Propionibacterium acnes/enzimología , Conformación Proteica , Alineación de SecuenciaRESUMEN
Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of ß-ketoacyl-acyl carrier protein synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate-binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Vibrio cholerae/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Acetilcoenzima A/metabolismo , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Antibacterianos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocombustibles , Cristalografía por Rayos X , Cisteína/genética , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Originally annotated as the initiator of fatty acid synthesis (FAS), ß-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Acinetobacter baumannii/enzimología , Secuencia de Aminoácidos , Ácidos Grasos/biosíntesis , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidad , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cisteína/química , Cisteína/metabolismo , Modelos Moleculares , Conformación Proteica , Especificidad por Sustrato , Difracción de Rayos XRESUMEN
We use mass spectrometry analysis and molecular modelling to show the established antimicrobial inhibitor 4,5-dichloro-1,2-dithiol-3-one (HR45) acts by forming a covalent adduct with the target ß-ketoacyl-ACP synthase III (FabH). The 5-chloro substituent directs attack of the essential active site thiol (C112) via a Michael-type addition elimination reaction mechanism.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Antiinfecciosos/farmacología , Inhibidores Enzimáticos/farmacología , Compuestos de Sulfhidrilo/farmacología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Modelos Moleculares , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the ß-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes, providing an excellent platform for the rational design of novel inhibitors. By comparing the structure of L. monocytogenes FabF with other published bacterial FabF structures in complex with known inhibitors and substrates, we highlight conformational changes within the active site, which will need to be accounted for during drug design and virtual screening studies. This high-resolution structure of FabF represents an important step in the development of new classes of antimicrobial agents targeting FabF for the treatment of listeriosis.