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1.
Sci Rep ; 10(1): 17776, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082446

RESUMO

Fatty acids are essential to most organisms and are made endogenously by the fatty acid synthase (FAS). FAS is an attractive target for antibiotics and many inhibitors are in clinical development. However, some gram-negative bacteria harbor an enzyme known as the acyl-acyl carrier protein synthetase (AasS), which allows them to scavenge fatty acids from the environment and shuttle them into FAS and ultimately lipids. The ability of AasS to recycle fatty acids may help pathogenic gram-negative bacteria circumvent FAS inhibition. We therefore set out to design and synthesize an inhibitor of AasS and test its effectiveness on an AasS enzyme from Vibrio harveyi, the most well studied AasS to date, and from Vibrio cholerae, a pathogenic model. The inhibitor C10-AMS [5'-O-(N-decanylsulfamoyl)adenosine], which mimics the tightly bound acyl-AMP reaction intermediate, was able to effectively inhibit AasS catalytic activity in vitro. Additionally, C10-AMS stopped the ability of Vibrio cholerae to recycle fatty acids from media and survive when its endogenous FAS was inhibited with cerulenin. C10-AMS can be used to study fatty acid recycling in other bacteria as more AasS enzymes continue to be annotated and provides a platform for potential antibiotic development.


Assuntos
Adenosina/síntese química , Antibacterianos/síntese química , Carbono-Enxofre Ligases/metabolismo , Cólera/microbiologia , Ácidos Graxos/metabolismo , Vibrio cholerae/fisiologia , Vibrio/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Antibacterianos/farmacologia , Catálise , Cólera/tratamento farmacológico , Desenvolvimento de Medicamentos , Ácido Graxo Sintases/metabolismo , Humanos , Especificidade por Substrato
2.
Mol Microbiol ; 113(4): 807-825, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31876062

RESUMO

Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anerobic, Gram-negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced the saturated fatty acids. The most abundant lipids were phosphatidylethanolamine (PE) and sulfonolipid (SL). Neither phosphatidylglycerol nor cardiolipin are present. PE synthesis is initiated by the PlsX/PlsY/PlsC pathway, whereas the SL pathway is related to sphingolipid biosynthesis. A. finegoldii incorporated medium-chain fatty acids (≤14 carbons) into PE and SL after their elongation, whereas long-chain fatty acids (≥16 carbons) were not elongated. Fatty acids >16 carbons were primarily incorporated into the 2-position of phosphatidylethanolamine at the PlsC step, the only biosynthetic enzyme that utilizes long-chain acyl-ACP. The ability to assimilate a broad-spectrum of fatty acid chain lengths present in the gut environment is due to the expression of two acyl-acyl carrier protein (ACP) synthetases. Acyl-ACP synthetase 1 had a substrate preference for medium-chain fatty acids and synthetase 2 had a substrate preference for long-chain fatty acids. This unique combination of synthetases allows A. finegoldii to utilize both the medium- and long-chain fatty acid nutrients available in the gut environment to assemble its membrane lipids.


Assuntos
Bacteroidetes/metabolismo , Ácidos Graxos/metabolismo , Microbioma Gastrointestinal , Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/metabolismo , Humanos , Lipídeos/biossíntese , Fosfatidiletanolaminas/biossíntese
3.
Bioorg Med Chem Lett ; 29(16): 2076-2078, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31300341

RESUMO

Mitomycins, produced by several Streptomyces strains, are potent anticancer antibiotics that comprise an aziridine ring fused to a tricyclic mitosane core. Mitomycins have remarkable ability to crosslink DNA with high efficiency. Despite long clinical history of mitomycin C, the biosynthesis of mitomycins, especially mitosane core formation, remains unknown. Here, we report in vitro characterization of three proteins, MmcB (acyl carrier protein), MitE (acyl AMP ligase), and MitB (glycosyltransferase) involved in mitosane core formation. We show that 3-amino-5-hydroxybenzoic acid (AHBA) is first loaded onto MmcB by MitE at the expense of ATP. MitB then catalyzes glycosylation of AHBA-MmcB with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to generate a key intermediate, GlcNAc-AHBA-MmcB, which contains all carbon and nitrogen atoms of the mitosane core. These results provide important insight into mitomycin biosynthesis.


Assuntos
Proteína de Transporte de Acila/química , Antibióticos Antineoplásicos/química , Proteínas de Bactérias/química , Carbono-Enxofre Ligases/química , Glicosiltransferases/química , Mitomicinas/biossíntese , Aminobenzoatos/química , Biocatálise , Hidroxibenzoatos/química , Mitomicinas/química , Streptomyces/enzimologia
4.
Biochemistry ; 58(25): 2804-2808, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31188570

RESUMO

Mitomycins make up a group of antitumor natural products that are biosynthesized from aminohydroxybenzoic acid (AHBA) and N-acetylglucosamine (GlcNAc). While the biosynthetic gene cluster was reported two decades ago, the mechanism by which the two building blocks, AHBA and GlcNAc, are coupled during biosynthesis remained uncharacterized. Here we report evidence that AHBA is first loaded onto an MmcB acyl carrier protein (ACP) by a MitE acyl ACP synthetase, followed by a transfer of GlcNAc from UDP-GlcNAc by MitB. The results suggest that the early steps of mitomycin biosynthesis proceed via intermediates linked to MmcB.


Assuntos
Proteína de Transporte de Acila/química , Carbono-Enxofre Ligases/química , Mitomicina/biossíntese , N-Acetilglucosaminiltransferases/química , Aminobenzoatos/química , Ensaios Enzimáticos , Hidroxibenzoatos/química , Cinética , Streptomyces/enzimologia
5.
Chembiochem ; 19(13): 1391-1395, 2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29603548

RESUMO

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although ß-amino acid loading in the fluvirucin B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the ß-amino acid loading pathway in fluvirucin B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer ß-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of ß-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.


Assuntos
Aminoácidos/metabolismo , Desoxiaçúcares/biossíntese , Actinobacteria/química , Actinobacteria/enzimologia , Aciltransferases/química , Aciltransferases/metabolismo , Aminoacilação , Carbono-Enxofre Ligases/química , Carbono-Enxofre Ligases/metabolismo , Carboxiliases/química , Carboxiliases/metabolismo , Catálise , Lactamas , Estrutura Molecular , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Domínios Proteicos , Especificidade por Substrato
6.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(11): 1404-1413, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27956138

RESUMO

Lysophospholipids (LPLs) are metabolic intermediates in bacterial phospholipid turnover. Distinct from their diacyl counterparts, these inverted cone-shaped molecules share physical characteristics of detergents, enabling modification of local membrane properties such as curvature. The functions of LPLs as cellular growth factors or potent lipid mediators have been extensively demonstrated in eukaryotic cells but are still undefined in bacteria. In the envelope of Gram-negative bacteria, LPLs are derived from multiple endogenous and exogenous sources. Although several flippases that move non-glycerophospholipids across the bacterial inner membrane were characterized, lysophospholipid transporter LplT appears to be the first example of a bacterial protein capable of facilitating rapid retrograde translocation of lyso forms of glycerophospholipids across the cytoplasmic membrane in Gram-negative bacteria. LplT transports lyso forms of the three bacterial membrane phospholipids with comparable efficiency, but excludes other lysolipid species. Once a LPL is flipped by LplT to the cytoplasmic side of the inner membrane, its diacyl form is effectively regenerated by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase (Aas). LplT-Aas also mediates a novel cardiolipin remodeling by converting its two lyso derivatives, diacyl or deacylated cardiolipin, to a triacyl form. This coupled remodeling system provides a unique bacterial membrane phospholipid repair mechanism. Strict selectivity of LplT for lyso lipids allows this system to fulfill efficient lipid repair in an environment containing mostly diacyl phospholipids. A rocker-switch model engaged by a pair of symmetric ion-locks may facilitate alternating substrate access to drive LPL flipping into bacterial cells. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.


Assuntos
Parede Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Lipogênese , Lisofosfolipídeos/biossíntese , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico , Carbono-Enxofre Ligases/química , Carbono-Enxofre Ligases/metabolismo , Lisofosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/química , Transdução de Sinais , Especificidade por Substrato
7.
Appl Microbiol Biotechnol ; 100(23): 10107-10113, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27704180

RESUMO

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.


Assuntos
Carbono-Enxofre Ligases/deficiência , Deleção de Genes , Synechococcus/enzimologia , Synechocystis/enzimologia , Farmacorresistência Bacteriana , Ácidos Láuricos/toxicidade , Ácido Linoleico/toxicidade , Mutação , Seleção Genética , Synechococcus/efeitos dos fármacos , Synechococcus/genética , Synechocystis/efeitos dos fármacos , Synechocystis/genética
8.
Sci Rep ; 6: 29076, 2016 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-27357109

RESUMO

Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. BCG comprises a number of substrains that exhibit genetic and biochemical differences. Whether and how these differences affect BCG efficacy remain unknown. Compared to other BCG strains, BCG-Japan, -Moreau, and -Glaxo are defective in the production of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), two lipid virulence factors. To determine if the loss of PDIMs/PGLs affects BCG efficacy, we constructed a PDIM/PGL-deficient strain of BCG-Pasteur by deleting fadD28, and compared virulence, immunogenicity, and protective efficacy in animal models. SCID mouse infection experiments showed that ∆fadD28 was more attenuated than wild type (WT). The ∆fadD28 and WT strains induced equivalent levels of antigen specific IFN-γ by CD4(+) and CD8(+) T cells; however, ∆fadD28 was less effective against Mycobacterium tuberculosis challenge in both BALB/c mice and guinea pigs. These results indicate that the loss of PIDMs/PGLs reduces the virulence and protective efficacy of BCG. Since the loss of PDIMs/PGLs occurs naturally in a subset of BCG strains, it also suggests that these strains may have been over-attenuated, which compromises their effectiveness. Our finding has important implications for current BCG programs and future vaccine development.


Assuntos
Vacina BCG/genética , Carbono-Enxofre Ligases/genética , Glicolipídeos/genética , Lipídeos/genética , Tuberculose/genética , Animais , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carbono-Enxofre Ligases/imunologia , Glicolipídeos/imunologia , Cobaias , Humanos , Japão , Lipídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Tuberculose/imunologia , Tuberculose/prevenção & controle , Fatores de Virulência/genética , Fatores de Virulência/imunologia
9.
J Biol Chem ; 291(15): 7973-89, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-26900152

RESUMO

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is one of the targets of first-line antituberculous drugs. This pathway contains a number of potential targets, including some that have been identified only recently and have yet to be explored. One such target, FadD32, is required for activation of the long meromycolic chain and is essential for mycobacterial growth. We report here an in-depth biochemical, biophysical, and structural characterization of four FadD32 orthologs, including the very homologous enzymes fromMycobacterium tuberculosisandMycobacterium marinum Determination of the structures of two complexes with alkyl adenylate inhibitors has provided direct information, with unprecedented detail, about the active site of the enzyme and the associated hydrophobic tunnel, shedding new light on structure-function relationships and inhibition mechanisms by alkyl adenylates and diarylated coumarins. This work should pave the way for the rational design of inhibitors of FadD32, a highly promising drug target.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Ligases/química , Ligases/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Carbono-Enxofre Ligases , Cristalografia por Raios X , Ligases/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium/química , Mycobacterium/efeitos dos fármacos , Infecções por Mycobacterium/tratamento farmacológico , Infecções por Mycobacterium/microbiologia , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Conformação Proteica , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
10.
PLoS One ; 10(12): e0145085, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26675168

RESUMO

Formaldehyde is a toxin and carcinogen that is both an environmental pollutant and an endogenous metabolite. Formaldehyde metabolism, which is probably essential for all aerobic cells, likely proceeds via multiple mechanisms, including via a glutathione-dependent pathway that is widely conserved in bacteria, plants and animals. However, it is unclear whether the first step in the glutathione-dependent pathway (i.e. formation of S-hydroxymethylglutathione (HMG)) is enzyme-catalysed. We report studies on glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans, which has been proposed to catalyse HMG formation from glutathione and formaldehyde on the basis of studies using NMR exchange spectroscopy (EXSY). Although we were able to replicate the EXSY results, time course experiments unexpectedly imply that GFA does not catalyse HMG formation under standard conditions. However, GFA was observed to bind glutathione using NMR and mass spectrometry. Overall, the results reveal that GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. Thus, it is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context.


Assuntos
Proteínas de Bactérias/química , Carbono-Enxofre Ligases/química , Paracoccus denitrificans/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/metabolismo , Glutationa/análogos & derivados , Glutationa/metabolismo , Dados de Sequência Molecular
11.
J Biol Chem ; 290(36): 22163-73, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26195634

RESUMO

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/metabolismo , Chlamydia trachomatis/metabolismo , Ácidos Graxos/metabolismo , Fosfolipídeos/biossíntese , Proteínas de Bactérias/genética , Carbono-Enxofre Ligases/genética , Chlamydia trachomatis/genética , Chlamydia trachomatis/fisiologia , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Cinética , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Especificidade por Substrato
12.
Plant Cell Physiol ; 56(8): 1608-15, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26063393

RESUMO

Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.


Assuntos
Carbono-Enxofre Ligases/metabolismo , Synechococcus/enzimologia , Aclimatação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/genética , Ácidos Graxos não Esterificados/metabolismo , Luz , Lipídeos de Membrana/metabolismo , Mutação , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/fisiologia , Complexo de Proteína do Fotossistema II/efeitos da radiação , Synechococcus/genética , Synechococcus/fisiologia , Synechococcus/efeitos da radiação
13.
Chem Biol ; 21(10): 1257-1259, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25373341

RESUMO

Acyl carrier proteins (ACPs) are promiscuous small proteins that play essential roles in the biosynthesis of many natural products, but our understanding of how they interact with various enzymes within larger protein complexes is lacking. In this issue of Chemistry and Biology, Beld and coworkers describe an enzymatic labeling method that will allow tracking of ACPs as they interact with their partners both in vitro and vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/metabolismo , Vibrio/enzimologia
14.
Chem Biol ; 21(10): 1293-1299, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25308274

RESUMO

The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/metabolismo , Vibrio/enzimologia , Arabidopsis/enzimologia , Proteínas de Bactérias/genética , Carbono-Enxofre Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato , Synechocystis/enzimologia , Thermus thermophilus/enzimologia
15.
Mol Microbiol ; 93(2): 262-75, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24866092

RESUMO

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.


Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Xanthomonas campestris/enzimologia , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Carbono-Enxofre Ligases/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/isolamento & purificação , Difusão , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Enzimas Multifuncionais/metabolismo , Família Multigênica , Mutação , Oxirredução , Doenças das Plantas/microbiologia , Transdução de Sinais/genética , Especificidade por Substrato , Tioléster Hidrolases/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/crescimento & desenvolvimento , Xanthomonas campestris/patogenicidade
16.
Metab Eng ; 24: 97-106, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24831705

RESUMO

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate.


Assuntos
Ésteres/metabolismo , Ácidos Graxos/metabolismo , Engenharia Metabólica , Mycobacterium tuberculosis , Policetídeos/metabolismo , Aciltransferases/biossíntese , Aciltransferases/genética , Carbono-Enxofre Ligases/biossíntese , Carbono-Enxofre Ligases/genética , Escherichia coli/enzimologia , Escherichia coli/genética
17.
Org Lett ; 15(18): 4854-7, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-24016264

RESUMO

Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their C-S bonds are formed by oxidative coupling reactions catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between histidine and cysteine (1 → 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 → 4 conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.


Assuntos
Carbono-Enxofre Ligases/metabolismo , Ergotioneína/biossíntese , Metilistidinas/síntese química , Betaína/análogos & derivados , Betaína/química , Catálise , Cisteína/química , Ergotioneína/química , Ergotioneína/metabolismo , Histidina/análogos & derivados , Histidina/biossíntese , Histidina/química , Histidina/metabolismo , Metilistidinas/química , Metilistidinas/metabolismo , Estrutura Molecular , Oxirredução , Estereoisomerismo , Compostos de Sulfidrila/química
18.
FEBS Lett ; 587(7): 936-42, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23454211

RESUMO

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Carbono-Enxofre Ligases/genética , Ácidos Graxos/metabolismo , Sementes/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Carbono-Enxofre Ligases/metabolismo , Cuphea/enzimologia , Cuphea/genética , Ácidos Graxos/química , Mutação , Plantas Geneticamente Modificadas , Plastídeos/enzimologia , Plastídeos/genética , Sementes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Triglicerídeos/metabolismo
19.
J Basic Microbiol ; 53(10): 848-55, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23417914

RESUMO

Free fatty acids are typically activated by thioesterification processes and catalyzed by the fatty acyl-CoA synthetase or fatty acyl-ACP synthetase. However, the routes for fatty acid activation in cyanobacteria are not well understood. In this investigation, the slr1609 gene, which encodes the fatty acid activation enzyme, was cloned from Synechocystis sp. PCC6803. This gene was identified by heterologous expression and in vitro enzymatic activity analyses. Different from previous reports stating that free fatty acids are only activated through the fatty acyl-ACP synthetases encoded by these genes in cyanobacteria, this gene was also proven to possess a fatty acyl-CoA synthetase activity, by in vitro enzymatic activity analyses and in vivo complementation experiments. The protein Slr1609 is located in both the cell membrane and the cytosol of Synechocystis sp. PCC6803. The differences in the transcriptional profiles between the wild type and the slr1609 deletion mutant strain were evaluated using microarray analyses. These analyses indicated that 299 differentially expressed genes are involved in fatty acid metabolism, photosynthesis, carbon fixation, stress tolerance and other metabolic processes. Our experiments demonstrate the observed compositional changes in the unsaturated fatty acids from the membrane lipids of the slr1609 deletion mutant when shifted from 30 to 24 °C.


Assuntos
Proteínas de Bactérias/metabolismo , Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Enzimas Multifuncionais/metabolismo , Synechocystis/enzimologia , Synechocystis/genética , Proteínas de Bactérias/genética , Carbono-Enxofre Ligases/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligases/genética , Citosol/metabolismo , Ácidos Graxos/química , Ácidos Graxos Insaturados/metabolismo , Genes Bacterianos , Lipídeos de Membrana/química , Análise em Microsséries , Enzimas Multifuncionais/genética , Temperatura , Transcrição Gênica
20.
Proteins ; 81(7): 1232-44, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23444054

RESUMO

In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA(Ile)2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site.


Assuntos
Aminoacil-tRNA Sintetases/química , Proteínas de Bactérias/química , Carbono-Enxofre Ligases/química , Estrutura Terciária de Proteína , Thermus thermophilus/enzimologia , Tiouridina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/enzimologia , Modelos Moleculares , Mutação
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