RESUMEN
Nervonic acid (C24:1) is a very-long-chain fatty acid that plays an imperative role in human brain development and other health benefits. In plants, 3-ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme for C24:1 biosynthesis. Xanthoceras sorbifolium is a valuable oil-producing economic woody species with abundant C24:1 in seed oils, but the key KCS gene responsible for C24:1 accumulation remains unknown. In this work, a correlation analysis between the transcript profiles of KCS and dynamic change of C24:1 content in developing seeds of X. sorbifolium were conducted to screen out three members of KCS, namely XsKCS4, XsKCS7 and XsKCS8, potentially involved in C24:1 biosynthesis. Of which, the XsKCS7 was highly expressed in developing seeds, while XsKCS4 and XsKCS8 displayed the highest expression in fruits and flowers, respectively. Overexpression of XsKCS4, XsKCS7 and XsKCS8 in yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana indicated that only XsKCS7 possessed the ability to facilitate the biosynthesis of C24:1. These findings collectively suggested that XsKCS7 played a crucial role in specific regulation of C24:1 biosynthesis in X. sorbifolium seeds.
Asunto(s)
Ácidos Grasos Monoinsaturados , Proteínas de Plantas , Sapindaceae , Semillas , Semillas/genética , Semillas/metabolismo , Semillas/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sapindaceae/genética , Sapindaceae/metabolismo , Sapindaceae/enzimología , Sapindaceae/crecimiento & desarrollo , Ácidos Grasos Monoinsaturados/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Plantas Modificadas Genéticamente/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The cuticle, consisting of cuticular wax and cutin, is a lipid membrane that seals the plant surface against environmental stress. ß-Ketoacyl-CoA synthases (KCSs) are condensing enzymes catalyzing crucial reactions elongating hydrocarbon chains into precursors for various cuticular wax components. Although many KCS genes were well characterized in various species, the functions of the closely related Arabidopsis KCS3, KCS12, KCS19 enzymes remained unclear. Here, we found KCS3 preferentially expressed in growing organs, especially in guard cells. kcs3 mutants and kcs3kcs12 double mutants displayed sepal fusion phenotypes, suggesting defects in cuticle formation. The mutants had decreased amounts of wax components with relatively short hydrocarbon chains in the developing organs but increased levels of wax compounds in mature organs. In contrast, kcs19 mutants showed seed fusion phenotypes and altered chain length distributions in seed suberin. Taken together, our results show that KCS12 and KCS3 share redundant functions in flower development, while KCS19 is involved in seed coat formation. All three condensing enzymes are involved in the elongation of C>18 hydrocarbon chains in young, actively expanding tissues.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Flores/genética , Flores/enzimología , Flores/crecimiento & desarrollo , Flores/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Semillas/enzimología , Ceras/metabolismo , Mutación , Fenotipo , Lípidos , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismoRESUMEN
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
The condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) by ß-ketoacyl-ACP synthase III (KAS III, FabH) and decarboxylation of malonyl-ACP by malonyl-ACP decarboxylase are the two pathways that initiate bacterial fatty acid synthesis (FAS) in Escherichia coli. In addition to these two routes, we report that Pseudomonas putida F1 ß-ketoacyl-ACP synthase I (FabB), in addition to playing a key role in fatty acid elongation, also initiates FAS in vivo. We report that although two P. putida F1 fabH genes (PpfabH1 and PpfabH2) both encode functional KAS III enzymes, neither is essential for growth. PpFabH1 is a canonical KAS III similar to E. coli FabH whereas PpFabH2 catalyzes condensation of malonyl-ACP with short- and medium-chain length acyl-CoAs. Since these two KAS III enzymes are not essential for FAS in P. putida F1, we sought the P. putida initiation enzyme and unexpectedly found that it was FabB, the elongation enzyme of the oxygen-independent unsaturated fatty acid pathway. P. putida FabB decarboxylates malonyl-ACP and condenses the acetyl-ACP product with malonyl-ACP for initiation of FAS. These data show that P. putida FabB, unlike the paradigm E. coli FabB, can catalyze the initiation reaction in FAS.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Pseudomonas putida , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Proteína Transportadora de Acilo/metabolismo , Escherichia coli/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos , Glucógeno Sintasa , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMEN
Sansevieria trifasciata Prain holds great potential as a valuable asset in pharmaceutical development. In this study, our focus is to explore and assess the antibacterial activity of various components derived from this plant, including extracts, fractions, subfractions, and isolates, explicitly targeting two common bacteria: Escherichia coli and Streptococcus aureus. The isolated compound, identified as a derivative pyridone alkaloid (5-methyl-11-(2-oxopyridin-1(2H)-yl)undecaneperoxoicacid), demonstrates notable antibacterial effects. The extracts, fractions, subfractions, and isolates reveal significant bacterial growth reductions (p < 0.05). The minimum inhibitory concentration (MIC) values for Escherichia coli were 1.95 ppm, 3.9 ppm, 15.62 ppm, and 7.81 ppm, respectively, while the MIC values for Streptococcus aureus were 1.95 ppm, 1.95 ppm, 15.62 ppm, and 7.81 ppm, respectively. Computational analysis showed the isolates' interaction with key residues on the active site of ß-ketoacyl-ACP synthase from Escherichia coli and TyrRS from Streptococcus aureus. The findings indicate that the isolates exhibit a strong affinity for specific residues, including His333, Cys163, and Phe392 in ß-ketoacyl-ACP synthase, as well as Arg88, His117, Glu160, and Gln213 in TyrRS. Comparative energy calculations using MMPBSA demonstrate the isolates' favorable binding energy (-104,101 kJ/mol for ß-ketoacyl-ACP synthase and -81,060 kJ/mol for TyrRS) compared to ciprofloxacin. The elucidated antibacterial activity and molecular interactions of the isolates present valuable knowledge for future in vitro studies, facilitating the development of novel antibacterial agents targeting diverse bacterial strains.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Sansevieria , Antibacterianos/farmacología , Ciprofloxacina , Escherichia coli , Extractos Vegetales/farmacologíaRESUMEN
The plant cuticle, consisting of wax and cutin, is involved in adaptations to various environments. ß-Ketoacyl-CoA synthases (KCSs) usually serve as a component of the fatty acid elongation complex that participates in the production of very long-chain fatty acids and provides precursors for the synthesis of various lipids, including wax; however, we recently reported that KCS3 and KCS12 negatively regulate wax biosynthesis. In this current study, we observed that unlike KCS3-overexpressing (OE) lines, KCS12-OE lines had fused floral organs because of abnormal cuticle biosynthesis. This prompted us to compare the functions of KCS3 and KCS12 during cuticle formation. Mutation of KCS3 caused greater effects on wax production, whereas mutation of KCS12 exerted more severe effects on cutin synthesis. The double-mutant kcs3 kcs12 had significantly increased wax and cutin contents compared to either single-mutant, suggesting that KCS12 and KCS3 have additive effects on cuticle biosynthesis. Cuticle permeability was greater for the double-mutant than for the single mutants, which ultimately led to increased susceptibility to drought stress and floral-organ fusion. Taken together, our results demonstrate the regulatory roles of KCS3 and KCS12 during cuticle biosynthesis, and show that maintaining KCS3 and KCS12 expression at certain levels is essential for the formation of a functional cuticle layer.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ceras , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Permeabilidad , Ceras/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) SintasaRESUMEN
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
The synthesis of fatty acids, which are essential for the growth and survival of bacterial cells, is catalyzed by beta-keto acyl-ACP synthase I-III. Due to the significant differences between the bacterial ACP synthase enzyme and the mammalian enzyme, it may serve as a viable target for the development of potent anti-bacterial medications. In this study, a sophisticated molecular docking strategy was employed to target all three KAS enzymes. Initially, 1000 fluoroquinolone derivatives were obtained from PubChem database, along with the commonly used ciprofloxacin, and subjected to virtual screening against FabH, FabB, and FabF, respectively. Subsequently, molecular dynamics (MD) simulations were conducted to confirm the stability and reliability of the generated conformations. The compounds 155813629, 142486676, and 155567217 were found to exhibit potential molecular interactions against FabH, FabB, and FabF, respectively, with docking scores of -9.9, -8.9, and -9.9 kcal/mol. These scores outperformed the docking score of standard ciprofloxacin. Furthermore, MD simulations were used to assess the dynamic nature of molecular interactions in both physiological and dynamic settings. Throughout the simulated trajectory, all three complexes displayed favorable stability patterns. The findings of this investigation suggest that fluoroquinolone derivatives may serve as highly effective and selective inhibitors of the KAS enzyme.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Fluoroquinolonas , Simulación del Acoplamiento Molecular , Fluoroquinolonas/farmacología , Reproducibilidad de los Resultados , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Inhibidores Enzimáticos/farmacologíaRESUMEN
BACKGROUND: Heat shock protein 60 (HSP60) is essential for the folding and assembly of newly imported proteins to the mitochondria. HSP60 is overexpressed in most types of cancer, but its association with ovarian cancer is still in dispute. SKOV3 and OVCAR3 were used as experimental models after comparing the expression level of mitochondrial HSP60 in a normal human ovarian epithelial cell line and four ovarian cancer cell lines. RESULTS: Low HSPD1 (Heat Shock Protein Family D (HSP60) Member 1) expression was associated with unfavorable prognosis in ovarian cancer patients. Knockdown of HSPD1 significantly promoted the proliferation and migration of ovarian cancer cells. The differentially expressed proteins after HSPD1 knockdown were enriched in the lipoic acid (LA) biosynthesis and metabolism pathway, in which mitochondrial 3-oxoacyl-ACP synthase (OXSM) was the most downregulated protein and responsible for lipoic acid synthesis. HSP60 interacted with OXSM and overexpression of OXSM or LA treatment could reverse proliferation promotion mediated by HSPD1 knockdown. CONCLUSIONS: HSP60 interacted with OXSM and maintained its stability. Knockdown of HSPD1 could promote the proliferation and migration of SKOV3 and OVCAR3 via lowering the protein level of OXSM and LA synthesis.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Proliferación Celular , Chaperonina 60 , Neoplasias Ováricas , Ácido Tióctico , Femenino , Humanos , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular/genética , Chaperonina 60/genética , Chaperonina 60/metabolismo , Proteínas de Choque Térmico , Proteínas Mitocondriales/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ácido Tióctico/farmacologíaRESUMEN
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional ß-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Mutagénesis , Ácidos GrasosRESUMEN
Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl-S-FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP). We envision that crystal structures of FabH with substrate analogues can provide insight into the conformational changes and enzyme/substrate interactions underpinning the distinct reactions. Here, we synthesize acetyl/malonyl-CoA analogues with esters or amides in place of the thioester and characterize their stability and behavior as Escherichia coli FabH substrates or inhibitors to inform structural studies. We also characterize the analogues with mutant FabH C112Q that mimics the acyl-enzyme intermediate allowing dissection of the decarboxylation reaction. The acetyl- and malonyl-oxa(dethia)CoA analogues undergo extremely slow hydrolysis in the presence of FabH or the C112Q mutant. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogues were completely stable to enzyme activity. In enzyme assays with acetyl-CoA and malonyl-CoA (rather than malonyl-ACP) as substrates, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki's near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH, including hysteresis for malonyl-CoA decarboxylation. The stability and inhibitory properties of the substrate analogues make them promising for structure-function studies to reveal fatty acid and polyketide enzyme/substrate interactions.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Policétidos , Acetilcoenzima A/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Proteína Transportadora de Acilo/química , Malonil Coenzima A/metabolismo , Ácidos GrasosRESUMEN
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
Fatty acid biosynthesis is essential for bacterial survival. Of these promising targets, ß-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target. A series of novel 1,3,4-oxadiazole-2(3H)-thione derivatives containing 1,4-benzodioxane skeleton targeting FabH were designed and synthesized. These compounds were determined by 1 H-NMR, 13 C-NMR, MS and further confirmed by crystallographic diffraction study for compound 7m and 7n. Most of the compounds exhibited good inhibitory activity against bacteria by computer-assisted screening, antibacterial activity test and E. coli FabH inhibitory activity test, wherein compounds 7e and 7q exhibited the most significant inhibitory activities. Besides, compound 7q showed the best E. coli FabH inhibitory activity (IC50 =2.45â µΜ). Computational docking studies also showed that compound 7q interacts with FabH critical residues in the active site.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Proteínas de Escherichia coli , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Antibacterianos/farmacología , Bacterias , Inhibidores Enzimáticos/química , Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Esqueleto/metabolismo , TionasRESUMEN
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
Chlorothricin (CHL) belongs to a spirotetronate antibiotic family produced by Streptomyces antibioticus that inhibits pyruvate carboxylase and malate dehydrogenase. For the biosynthesis of CHL, ChlB3 plays a crucial role by introducing the 6-methylsalicylic acid (6MSA) moiety to ChlB2, an acyl carrier protein (ACP). However, the structural insight and catalytic mechanism of ChlB3 was unclear. In the current study, the crystal structure of ChlB3 was solved at 3.1 Å-resolution and a catalytic mechanism was proposed on the basis of conserved residues of structurally related enzymes. ChlB3 is a dimer having the same active sites as CerJ (a structural homologous enzyme) and uses a KSIII-like fold to work as an acyltransferase. The relaxed substrate specificity of ChlB3 was defined by its catalytic efficiencies (kcat/Km) for non-ACP tethered synthetic substrates such as 6MSA-SNAC, acetyl-SNAC, and cyclohexonyl-SNAC. ChlB3 successfully detached the 6MSA moiety from 6MSA-SNAC substrate and this hydrolytic activity demonstrated that ChlB3 has the potential to catalyze non-ACP tethered substrates. Structural comparison indicated that ChlB3 belongs to FabH family and showed 0.6-2.5 Å root mean square deviation (RMSD) with structural homologous enzymes. Molecular docking and dynamics simulations were implemented to understand substrate active site and structural behavior such as the open and closed conformation of the ChlB3 protein. The resultant catalytic and substrate recognition mechanism suggested that ChlB3 has the potential to use non-native substrates and minimize the labor of expressing ACP protein. This versatile acyltransferase activity may pave the way for manufacturing CHL variants and may help to hydrolyze several thioester-based compounds.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Proteína Transportadora de Acilo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Aminoglicósidos , Antibacterianos , Malato Deshidrogenasa/metabolismo , Simulación del Acoplamiento Molecular , Piruvato Carboxilasa/metabolismo , Especificidad por SustratoRESUMEN
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
Very long chain fatty acids (VLCFAs) are precursors to sphingolipids, glycerophospholipids, and plant cuticular waxes. In plants, members of a large 3-ketoacyl-CoA synthase (KCS) gene family catalyze the substrate-specific elongation of VLCFAs. Although it is well understood that KCSs have evolved to use diverse substrates, the underlying molecular determinants of their specificity are still unclear. In this study, we exploited the sequence similarity of a KCS gene cluster from Populus trichocarpa to examine the evolution and molecular determinants of KCS substrate specificity. Functional characterization of five members (PtKCS1, 2, 4, 8, 9) in yeast showed divergent product profiles based on VLCFA length, saturation, and position of the double bond. In addition, homology models, rationally designed chimeras, and site-directed mutants were used to identify two key regions (helix-4 and position 277) as being major determinants of substrate specificity. These results were corroborated with chimeras involving a more distantly related KCS, PtCER6 (the poplar ortholog of the Arabidopsis CER6), and used to show that helix-4 is necessary for the modulatory effect of PtCER2-like5 on KCS substrate specificity. The role of position 277 in limiting product length was further tested by substitution with smaller amino acids, which shifted specificity toward longer products. Finally, treatment with KCS inhibitors (K3 herbicides) showed varying inhibitor sensitivities between the duplicated paralogs despite their sequence similarity. Together, this work sheds light on the molecular mechanisms driving substrate diversification in the KCS family and lays the groundwork for tailoring the production of specific VLCFAs.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Arabidopsis , Populus , Especificidad por Sustrato , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Populus/genética , Populus/metabolismo , Ácidos Grasos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Familia de Multigenes , Plantas/metabolismo , Coenzima A/metabolismoRESUMEN
Fatty acid biosynthesis is essential for bacterial survival. Of these promising targets, ß-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target. FabH would trigger the initiation of fatty acid biosynthesis and it is highly conserved among Gram-positive and -negative bacteria. A series of novel amide derivatives bearing dioxygenated rings were synthesized and developed as potent inhibitors of FabH. These compounds were determined by 1H-NMR, 13C-NMR, MS and further confirmed by crystallographic diffraction study for compound 19. Furthermore, these compounds were evaluated strong broad-spectrum antibacterial activity. Some compounds with potent antibacterial activities were tested for their Escherichia coli (E. coli) FabH inhibitory activity. Especially, compound 19 showed the most potent antibacterial activity with minimum inhibitory concentration (MIC) values of 1.56-3.13 mg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC50 of 2.4 µM. Docking simulation was performed to position compound 19 into the E. coli FabH active site to determine the probable binding conformation.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Proteínas de Escherichia coli , Amidas , Antibacterianos/química , Bacterias/metabolismo , Inhibidores Enzimáticos/química , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Ácidos Grasos , Transferasas (Grupos de Otros Fosfatos Sustitutos)RESUMEN
Since the early twentieth century, with the isolation of penicillin and streptomycin in the 1940s, the modern era of anti-infective drug development has gained momentum. Due to the enormous success of early drug discovery, many infectious diseases were successfully prevented and eradicated. However, this initial hope was wrongheaded, and pathogens evolved as a significant threat to human health. Drug resistance develops as a result of natural selection's relentless pressure, necessitating the identification of new drug targets and the creation of chemotherapeutics that bypass existing drug resistance mechanisms. Fatty acid biosynthesis (FAS) is a crucial metabolic mechanism for bacteria during their growth and development. Several crucial enzymes involved in this biosynthetic pathway have been identified as potential targets for new antibacterial agents. In Escherichia coli (E. coli), this pathway has been extensively investigated. The present review focuses on progress in the development of Kas A, Kas B, and Fab H inhibitors as mono-therapeutic antibiotics.