RESUMEN
Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the ß-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Pared Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/biosíntesis , Shewanella/metabolismo , Proteína Transportadora de Acilo/genética , Pared Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/genética , Shewanella/genéticaRESUMEN
Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.
Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Biotina/biosíntesis , Cadaverina/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Transaminasas/metabolismo , Acetil-CoA Carboxilasa/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Fenotipo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Transaminasas/genéticaRESUMEN
Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Acido Graso Sintasa Tipo II/química , Ácidos Grasos/química , Hidroliasas/química , Simulación de Dinámica Molecular , Complejos Multienzimáticos/química , Proteína Transportadora de Acilo/genética , Proteína Transportadora de Acilo/metabolismo , Catálisis , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Hidroliasas/genética , Hidroliasas/metabolismo , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismoRESUMEN
The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
Asunto(s)
Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/biosíntesis , Photobacterium/genética , Escherichia coli/genética , Acido Graso Sintasa Tipo II/metabolismo , Mutación , Photobacterium/metabolismoRESUMEN
Elongating ketosynthases (KSs) catalyze carbon-carbon bond-forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, the ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution details of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region deletion variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of an entire region significantly compromising activity. Structure and sequence analyses of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.
Asunto(s)
Acetiltransferasas/metabolismo , Proteína Transportadora de Acilo/metabolismo , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/biosíntesis , Acetiltransferasas/genética , Proteína Transportadora de Acilo/genética , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/genética , Mutagénesis Sitio-Dirigida , Mutación PuntualRESUMEN
Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Vías Biosintéticas , Dominio Catalítico/genética , Cristalografía por Rayos X , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos Insaturados/química , Humanos , Espectrometría de Masas , Modelos Moleculares , Moritella/enzimología , Moritella/genética , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Especificidad por SustratoRESUMEN
Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host. A previous developed switch for butenoic acid fermentation is based on triclosan molecule as the FabI inhibitor in the fatty acid biosynthesis cycle. However, triclosan is toxic to human, which may limit its pharmaceutical application. Alternatively, we here purposed a nontoxic switch of carbon flux by harnessing recently developed CRISPR interference (CRISPRi) approach. In our work, we constructed a CRISPRi/dCpf1-mediated dynamic metabolic switch to separate the host growth and production phase via switching the expression of the fabI gene in fatty acid biosynthesis pathway. After optimizing the programmable targets, the CRISPRi-based switch boosted the titer of butenoic acid by 6-fold (1.41 g/L) in fed-batch fermentation. Our work supported that the CRISPRi/dCpf1 switch could replace triclosan-based switch as a nontoxic switch for butenoic acid production, and outcompeted the later switch in the biomass accumulation of the host cell. Moreover, the CRISPRi/dCpf1 system was integrated into the chromosome of the host to improve its genetic stability for long-term fermentation and other applications.Key Points⢠A programmable metabolic switch was developed to replace the toxic chemical switch to separate the growth phase and production phase of the butenoic acid.⢠The programmable CRISPRi/dCpf1 switch was efficiently and stably integrated into the host genome to increase their genetic stability during fermentation.⢠The optimized metabolic switch simultaneously increased the host biomass and butenoic acid titer, and solved the paradox of the competition between growth and production.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Ingeniería Metabólica , Técnicas de Cultivo Celular por Lotes , Biomasa , Vías Biosintéticas , Ciclo del Carbono , Enoil-ACP Reductasa (NADH)/genética , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/genética , Fermentación , Genoma Bacteriano , Microbiología IndustrialRESUMEN
Type II fatty acid biosynthesis in bacteria can be broadly classified into the initiation and elongation phases. The biochemical functions defining each step in the two phases have been studied in vitro Among the ß-ketoacyl-acyl carrier protein (ACP) synthases, FabH catalyzes the initiation reaction, while FabB and FabF, which primarily catalyze the elongation reaction, can also drive initiation as side reactions. A role for FabB and FabF in the initiation of fatty acid biosynthesis would be supported by the viability of the ΔfabH mutant. In this study, we show that the ΔfabH and ΔyiiD mutations were synthetically lethal and that ΔfabH ΔrelA ΔspoT and ΔfabH ΔdksA synthetic lethality was rescued by the heterologous expression of yiiD In the ΔfabH mutant, the expression of yiiD was positively regulated by (p)ppGpp. The growth defect, reduced cell size, and altered fatty acid profile of the ΔfabH mutant and the growth defect of the ΔfabH ΔfabF fabB15(Ts) mutant in oleate- and palmitate-supplemented medium at 42°C were rescued by the expression of yiiD from a multicopy plasmid. Together, these results indicate that the yiiD-encoded function supported initiation of fatty acid biosynthesis in the absence of FabH. We have renamed yiiD as fabYIMPORTANCE Fatty acid biosynthesis is an essential process conserved across life forms. ß-Ketoacyl-ACP synthases are essential for fatty acid biosynthesis. FabH is a ß-ketoacyl-ACP synthase that contributes to the initiation of fatty acid biosynthesis in Escherichia coli In this study, we present genetic and biochemical evidence that the yiiD (renamed fabY)-encoded function contributes to the biosynthesis of fatty acid in the absence of FabH activity and that under these conditions, the expression of FabY was regulated by the stringent response factors (p)ppGpp and DksA. Combined inactivation of FabH and FabY resulted in growth arrest, possibly due to the loss of fatty acid biosynthesis. A molecule(s) that inhibits the two activities can be an effective microbicide.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ácidos Grasos/biosíntesis , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/genética , GTP Pirofosfoquinasa/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato/metabolismo , Mutación , Mutaciones Letales SintéticasRESUMEN
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) using a (CO2)6k+ gas cluster ion beam (GCIB) was used to analyze Escherichia coli mutants previously identified as having impaired plasmid transfer capability related to the spread of antibiotic resistance. The subset of mutants selected were expected to result in changes in the bacterial envelope composition through the deletion of genes encoding for FabF, DapF, and Lpp, where the surface sensitivity of ToF-SIMS can be most useful. Analysis of arrays of spotted bacteria allowed changes in the lipid composition of the bacteria to be elucidated using multivariate analysis and confirmed through imaging of individual ion signals. Significant changes in chemical composition were observed, including a surprising loss of cyclopropanated fatty acids in the fabF mutant where FabF is associated with the elongation of FA(16:1) to FA(18:1) and not cyclopropane formation. The ability of the GCIB to generate increased higher mass signals from biological samples allowed intact lipid A (m/z 1796) to be detected on the bacteria and, despite a 40 keV impact energy, depth profiled through the bacterial envelope along with other high mass ions including species at m/z 1820 and 2428, attributed to ECACYC, that were only observed below the surface of the bacteria and were notably absent in the depth profile of the lpp mutant. The analysis provides new insights into the action of the specific pathways targeted in this study and paves the way for whole new avenues for the characterization of intact molecules within the bacterial envelope.
Asunto(s)
Membrana Celular/química , Escherichia coli/genética , Plásmidos/metabolismo , Espectrometría de Masa de Ion Secundario/métodos , Acetiltransferasas/genética , Isomerasas de Aminoácido/genética , Proteínas de la Membrana Bacteriana Externa/genética , Farmacorresistencia Microbiana , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/análisis , Lípido A/análisis , Lípidos/análisis , Lipoproteínas/genética , Proteínas MutantesRESUMEN
De novo biosynthesis of fatty acids is an iterative process requiring strict regulation of the lengths of the produced fatty acids. In this review, we focus on the factors determining chain lengths in fatty acid biosynthesis. In a nutshell, the process of chain-length regulation can be understood as the output of a chain-elongating C-C bond forming reaction competing with a terminating fatty acid release function. At the end of each cycle in the iterative process, the synthesizing enzymes need to "decide" whether the growing chain is to be elongated through another cycle or released as the "mature" fatty acid. Recent research has shed light on the factors determining fatty acid chain length and has also achieved control over chain length for the production of the technologically interesting short-chain (C4 -C8 ) and medium-chain (C10 -C14 ) fatty acids.
Asunto(s)
Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo I/química , Ácidos Grasos/biosíntesis , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Animales , Bacterias/enzimología , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/química , Humanos , Estructura Molecular , Plantas/enzimología , Dominios Proteicos , Ingeniería de Proteínas , Saccharomyces cerevisiae/enzimologíaRESUMEN
DNA vaccines require a vector to replicate genes and express encoding antigens. Antibiotic resistance genes are often used as selection markers, which must not be released to the environment upon final product commercialization. For this reason, generation of antibiotic resistance-free vectors is imperative. The pPAL vector contains the cytomegalovirus enhancer and promoter for expression in mammalian cells and the E. coli fabI chromosomal gene as a selectable marker. The fabI gene encodes the enoyl-ACP reductase (FabI). The bacteriostatic compound triclosan is an inhibitor of this enzyme. Therefore, the selection of positive clones depends on the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding for the protein kinase C receptor analog (LACK/p36) in the HEK293T human cell line transfected with pPAL-LACK. The fabI gene sequence contains a 210 bp CpG island, suggesting a potential role as an adjuvant of the antibiotic resistance-free pPAL vector. In fact, Th1 response induction levels against canine leishmaniasis only using pPAL-LACK was shown to be as strong as in previous strategies using a recombinant vaccinia virus in combination with standard mammalian expression plasmid vectors. In summary, the pPAL plasmid contains the essential elements for manipulation and expression of any cloned DNA sequence in prokaryotic and mammalian cells using an E. coli endogenous gene as a selectable marker, which also provides a long CpG island. This element enhances Th1 immune response against L. infantum infection in dogs using the gene encoding for the LACK antigen. Therefore, this antibiotic resistance-free plasmid is a vaccine vector actively participating in protection against canine leishmaniasis and may be potentially tested as a vaccine vector with other antigens against different pathogens.
Asunto(s)
Antígenos de Protozoos/genética , Leishmania infantum/efectos de los fármacos , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/prevención & control , Plásmidos/inmunología , Proteínas Protozoarias/genética , Vacunas de ADN/inmunología , Animales , Antígenos de Protozoos/inmunología , Islas de CpG , Citomegalovirus/genética , Perros , Farmacorresistencia Microbiana , Elementos de Facilitación Genéticos , Enoil-ACP Reductasa (NADH)/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/genética , Marcadores Genéticos , Células HEK293 , Humanos , Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/administración & dosificación , Vacunas contra la Leishmaniasis/genética , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Plásmidos/administración & dosificación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Proteínas Protozoarias/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/parasitología , Triclosán/farmacología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
OBJECTIVE: To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. RESULTS: Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 µM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. CONCLUSION: H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Bacterianas/química , Simulación por Computador , Acido Graso Sintasa Tipo II/química , Hemina/química , Rhodopseudomonas/enzimología , Análisis de Secuencia de Proteína , Proteínas Bacterianas/genética , Estabilidad de Enzimas/genética , Acido Graso Sintasa Tipo II/genética , Calor , Rhodopseudomonas/genéticaRESUMEN
The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.
Asunto(s)
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Lipopolisacáridos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Transferasas/fisiología , Proteasas ATP-Dependientes/deficiencia , Proteasas ATP-Dependientes/genética , Acetiltransferasas/deficiencia , Acetiltransferasas/genética , Amidohidrolasas/fisiología , Catálisis , Biología Computacional , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/deficiencia , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos Insaturados/metabolismo , Regulación Bacteriana de la Expresión Génica , Heptosas/biosíntesis , Lípido A/biosíntesis , Redes y Vías Metabólicas/fisiología , Modelos Biológicos , Biogénesis de Organelos , Ácido Palmítico/farmacología , Azúcares Ácidos/metabolismo , Transferasas/biosíntesis , Transferasas/genéticaRESUMEN
Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles. The former are considered primary metabolites that are linear hydrocarbon acids, while the latter are complex aromatic or polyunsaturated secondary metabolites. Though the study of bacterial FAS has benefitted from decades of biochemical and structural investigations, type II PKSs have remained less understood. Here we review the recent approaches to understanding the protein-protein and protein-substrate interactions in these pathways, with an emphasis on recent chemical biology and structural applications. New approaches to the study of FAS have highlighted the critical role of the acyl carrier protein (ACP) with regard to how it stabilizes intermediates through sequestration and selectively delivers cargo to successive enzymes within these iterative pathways, utilizing protein-protein interactions to guide and organize enzymatic timing and specificity. Recent tools that have shown promise in FAS elucidation should find new approaches to studying type II PKS systems in the coming years.
Asunto(s)
Acido Graso Sintasa Tipo II/metabolismo , Sintasas Poliquetidas/metabolismo , Mapas de Interacción de Proteínas , Cristalografía por Rayos X , Ciclización , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/genética , Helicobacter pylori/metabolismo , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Proteínas/análisis , Proteínas/química , Proteínas/genética , Especificidad por SustratoRESUMEN
Bacterial fatty acid synthesis (FAS) has been extensively studied as a potential target of antimicrobials. In FAS, FabD mediates transacylation of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP). The mounting threat of nosocomial infection by multidrug-resistant Acinetobacter baumannii warrants a deeper understanding of its essential cellular mechanisms, which could lead to effective control of this highly competent pathogen. The molecular mechanisms involved in A. baumannii FAS are poorly understood, and recent research has suggested that Pseudomonas aeruginosa, a closely related nosocomial pathogen of A. baumannii, utilizes FAS to produce virulence factors. In this study, we solved the crystal structure of A. baumannii FabD (AbFabD) to provide a platform for the development of new antibacterial agents. Analysis of the structure of AbFabD confirmed the presence of highly conserved active site residues among bacterial homologs. Binding constants between AbFabD variants and A. baumannii ACP (AbACP) revealed critical conserved residues Lys195 and Lys200 involved in AbACP binding. Computational docking of a potential inhibitor, trifluoperazine, revealed a unique inhibitor-binding pocket near the substrate-binding site. The structural study presented herein will be useful for the structure-based design of potent AbFabD inhibitors.
Asunto(s)
Acinetobacter baumannii/genética , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/genética , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Acido Graso Sintasa Tipo II/genética , Acinetobacter baumannii/enzimología , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/química , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/metabolismo , Modelos Moleculares , Mutación , Dominios Proteicos , Homología de Secuencia de AminoácidoRESUMEN
Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Medios de Cultivo/química , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crecimiento & desarrollo , Escherichia coli/genética , MutaciónRESUMEN
We report the characterization of the Arabidopsis (Arabidopsis thaliana) 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA sequencing analysis revealed that mthd-rnai and mtkas mutants are nearly equivalent to each other in altering the transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Acido Graso Sintasa Tipo II/metabolismo , Ácido Graso Sintasas/metabolismo , Hidroliasas/metabolismo , Mitocondrias/enzimología , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Western Blotting , Dióxido de Carbono/metabolismo , Acido Graso Sintasa Tipo II/genética , Ácido Graso Sintasas/genética , Regulación de la Expresión Génica de las Plantas , Glicolatos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidroliasas/genética , Metabolómica/métodos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Mutación , Ácidos Mirísticos/metabolismo , Plantas Modificadas Genéticamente , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN/métodos , Homología de Secuencia de Aminoácido , Sacarosa/metabolismoRESUMEN
The enzyme acetyl-CoA carboxylase (ACCase) catalyzes the committed step of the de novo fatty acid biosynthesis (FAS) pathway by converting acetyl-CoA to malonyl-CoA. Two forms of ACCase exist in nature, a homomeric and heteromic form. The heteromeric form of this enzyme requires four different subunits for activity: biotin carboxylase; biotin carboxyl carrier protein; and α- and ß-carboxyltransferases. Heteromeric ACCases (htACCase) can be found in prokaryotes and the plastids of most plants. The plant htACCase is regulated by diverse mechanisms reflected by the biochemical and genetic complexity of this multienzyme complex and the plastid stroma where it resides. In this review we summarize the regulation of the plant htACCase and also describe the structural characteristics of this complex from both prokaryotes and plants. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Ligasas de Carbono-Nitrógeno/genética , Ácidos Grasos/biosíntesis , Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilasa/química , Secuencia de Aminoácidos/genética , Ligasas de Carbono-Nitrógeno/química , Transferasas de Carboxilo y Carbamoilo/química , Transferasas de Carboxilo y Carbamoilo/genética , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos/genética , Plantas/enzimología , Plastidios/enzimología , Células Procariotas/enzimologíaRESUMEN
The type II fatty acid synthesis (FASII) pathway is essential for bacterial lipid biosynthesis and continues to be a promising target for novel antibacterial compounds. Recently, it has been demonstrated that Chlamydia is capable of FASII and this pathway is indispensable for Chlamydia growth. Previously, a high-content screen with Chlamydia trachomatis-infected cells was performed, and acylated sulfonamides were identified to be potent growth inhibitors of the bacteria. C. trachomatis strains resistant to acylated sulfonamides were isolated by serial passage of a wild-type strain in the presence of low compound concentrations. Results from whole-genome sequencing of 10 isolates from two independent drug-resistant populations revealed that mutations that accumulated in fabF were predominant. Studies of the interaction between the FabF protein and small molecules showed that acylated sulfonamides directly bind to recombinant FabF in vitro and treatment of C. trachomatis-infected HeLa cells with the compounds leads to a decrease in the synthesis of Chlamydia fatty acids. This work demonstrates the importance of FASII for Chlamydia development and may lead to the development of new antimicrobials.
Asunto(s)
Antibacterianos/farmacología , Chlamydia trachomatis/efectos de los fármacos , Acido Graso Sintasa Tipo II/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Ácidos Grasos/biosíntesis , Sulfametoxazol/farmacología , Acilación/efectos de los fármacos , Adamantano/farmacología , Aminobenzoatos/farmacología , Anilidas/farmacología , Animales , Línea Celular Tumoral , Cerulenina/farmacología , Infecciones por Chlamydia/tratamiento farmacológico , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlorocebus aethiops , Acido Graso Sintasa Tipo II/genética , Células HeLa , Humanos , Triclosán/farmacología , Células VeroRESUMEN
The need for new antimicrobials to treat bacterial infections has led to the use of type II fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to the screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, nonculturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in the acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of nonculturable variants.