RESUMEN
Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called "ideal" folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes.
Asunto(s)
S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Anticuerpos/química , Anticuerpos/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Insulina/metabolismo , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/inmunología , Anticuerpos/inmunología , Sitios de Unión de Anticuerpos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Insulina/inmunología , Ligandos , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Conformación ProteicaRESUMEN
Biomolecular recognition underlying drug-target interactions is determined by both binding affinity and specificity. Whilst, quantification of binding efficacy is possible, determining specificity remains a challenge, as it requires affinity data for multiple targets with the same ligand dataset. Thus, understanding the interaction space by mapping the target space to model its complementary chemical space through computational techniques are desirable. In this study, active site architecture of FabD drug target in two apicomplexan parasites viz. Plasmodium falciparum (PfFabD) and Toxoplasma gondii (TgFabD) is explored, followed by consensus docking calculations and identification of fifteen best hit compounds, most of which are found to be derivatives of natural products. Subsequently, machine learning techniques were applied on molecular descriptors of six FabD homologs and sixty ligands to induce distinct multivariate partial-least square models. The biological space of FabD mapped by the various chemical entities explain their interaction space in general. It also highlights the selective variations in FabD of apicomplexan parasites with that of the host. Furthermore, chemometric models revealed the principal chemical scaffolds in PfFabD and TgFabD as pyrrolidines and imidazoles, respectively, which render target specificity and improve binding affinity in combination with other functional descriptors conducive for the design and optimization of the leads.
Asunto(s)
S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Antimaláricos/química , Inhibidores Enzimáticos/química , Modelos Químicos , Plasmodium falciparum/enzimología , Proteínas Protozoarias , Toxoplasma/enzimología , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/antagonistas & inhibidores , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/química , Sistemas de Liberación de Medicamentos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/químicaAsunto(s)
Productos Biológicos/biosíntesis , Mercurio/metabolismo , Oxidorreductasas/metabolismo , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Oxidorreductasas/aislamiento & purificación , Pseudomonas aeruginosa/metabolismoRESUMEN
Polyketides are a class of natural products that exhibit a wide range of functional and structural diversity. They include antibiotics, immunosuppressants, antifungals, antihypercholesterolemics, and cytotoxins. Polyketide synthases (PKSs) use chemistry similar to fatty acid synthases (FASs), although building block variation and differing extents of reduction of the growing polyketide chain underlie their biosynthetic versatility. In contrast to the well studied sequential modular type I PKSs, less is known about how the iterative type I PKSs carry out and control chain initiation, elongation, folding, and cyclization during polyketide processing. Domain structure analysis of a group of related fungal, nonreducing PKSs has revealed well defined N-terminal domains longer than commonly seen for FASs and modular PKSs. Predicted structure of this domain disclosed a region similar to malonyl-CoA:acyl-carrier protein (ACP) transacylases (MATs). MATs play a key role transferring precursor CoA thioesters from solution onto FASs and PKSs for chain elongation. On the basis of site-directed mutagenesis, radiolabeling, and kinetics experiments carried out with individual domains of the norsolorinic acid PKS, we propose that the N-terminal domain is a starter unit:ACP transacylase (SAT domain) that selects a C(6) fatty acid from a dedicated yeast-like FAS and transfers this unit onto the PKS ACP, leading to the production of the aflatoxin precursor, norsolorinic acid. These findings could indicate a much broader role for SAT domains in starter unit selection among nonreducing iterative, fungal PKSs, and they provide a biochemical rationale for the classical acetyl "starter unit effect."
Asunto(s)
S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/química , Sintasas Poliquetidas/química , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/genética , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Aflatoxinas/biosíntesis , Aflatoxinas/química , Secuencia de Aminoácidos , Aspergillus/enzimología , Aspergillus/genética , Secuencia de Bases , Clonación Molecular , ADN de Hongos/genética , Genes Fúngicos , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Incubation of rat peritoneal macrophages in the presence of thapsigargin increased production of prostaglandin E2, intracellular platelet-activating factor (PAF) and interleukin-6. However, no PAF was detected in the conditioned medium. In the presence of SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydroimidazol-1-yl)heptane phosphonate), a CoA-independent transacylase inhibitor, the thapsigargin-induced increases in the interleukin-6 mRNA level and interleukin-6 production were suppressed in a concentration-dependent manner. This inhibitor also suppressed the production of prostaglandin E2 and intracellular PAF. The PAF receptor antagonists such as E6123 ((S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine) and L-652,731 (2,5-bis(3,4,5-trimethylphenyl)tetrahydrofuran) partially inhibited the thapsigargin-induced increase in the levels of interleukin-6 mRNA and interleukin-6 protein. The SK&F 98625-induced suppression of interleukin-6 mRNA accumulation and interleukin-6 production was partially restored by addition of exogenous prostaglandin E2. However, exogenous PAF failed to reverse the suppression suggesting that the intracellular PAF does not act in an autocrine mechanism. These findings suggested that the concurrently produced prostaglandin E2 and intracellular PAF participate in the thapsigargin-induced increase in the interleukin-6 mRNA level and interleukin-6 production by rat peritoneal macrophages.
Asunto(s)
Dinoprostona/fisiología , Interleucina-6/biosíntesis , Factor de Activación Plaquetaria/fisiología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Tapsigargina/farmacología , Acetiltransferasas/antagonistas & inhibidores , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Animales , Azepinas/farmacología , Células Cultivadas , Dinoprostona/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Furanos/farmacología , Imidazoles/farmacología , Indometacina/farmacología , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Compuestos Organofosforados/farmacología , Factor de Activación Plaquetaria/antagonistas & inhibidores , Factor de Activación Plaquetaria/biosíntesis , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/fisiología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Tapsigargina/antagonistas & inhibidores , Triazoles/farmacologíaRESUMEN
Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Acetiltransferasas/metabolismo , Streptomyces/enzimología , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/aislamiento & purificación , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Humanos , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tiofenos/farmacologíaRESUMEN
The enzyme coenzyme A-independent transacylase (CoA-IT) has been demonstrated to be the key mediator of arachidonate remodeling, a process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the release of arachidonate in stimulated neutrophils and inhibit the production of eicosanoids and platelet-activating factor. We describe novel inhibitors of CoA-IT activity that contain a beta-lactam nucleus. beta-Lactams were investigated as potential mechanism-based inhibitors of CoA-IT on the basis of the expected formation of an acyl-enzyme intermediate complex. Two beta-lactams, SB 212047 and SB 216754, were shown to be specific, time-dependent inhibitors of CoA-IT activity (IC50 = 6 and 20 microM, respectively, with a 10-min pretreatment time). Extensive washing and dilution could not remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a time-dependent manner. In an in vivo model of phorbol ester-induced ear inflammation, SB 216754 was able to inhibit indices of both edema and cell infiltration. Taken together, the results support two hypotheses: 1) CoA-IT activity is important for the production of inflammatory lipid mediators in stimulated cells and in vivo and 2) the mechanism by which CoA-IT acts to transfer arachidonate is through an acyl-enzyme intermediate.
Asunto(s)
Acetiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Lactamas , beta-Lactamas/farmacología , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Animales , Antiinflamatorios/farmacología , Línea Celular , Eicosanoides/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Microsomas/enzimología , Neutrófilos/enzimología , Factor de Activación Plaquetaria/metabolismoRESUMEN
It has been suggested that phospholipase A2 (PLA2) contributes to the regulation of leukotriene (LT) and platelet-activating factor (PAF) synthesis by controlling the release of their precursors, arachidonic acid (AA) and lysophosphatidylcholine (lysoPC), from membrane phospholipids. In rat alveolar macrophages (AMs), PLA2 appears to have a major role in LT synthesis but a more limited role in PAF synthesis. The present study was designed to define the role of PLA2 in LT and PAF synthesis in human AMs and determine whether differences exist between AMs obtained from normal subjects and those from patients with asthma. In the normal subjects, the calcium ionophore A23187 (Cal) increased AM PAF synthesis (percent incorporation of tritiated acetate) by 135% (p < 0.01) and LTB4 synthesis 88-fold (p < 0.001). Phorbol myristate acetate (PMA) had little effect alone, but it had a synergistic effect with Cal, increasing PAF synthesis by 466% and LTB4 synthesis to 229-fold above the control values (p < 0.001 for both). Ro 25-4331, a combined cytosolic (c) and secretory (s) PLA2 inhibitor, had little effect on the Cal-stimulated PAF synthesis, but it completely blocked the effect of PMA. It also blocked the Cal- and Cal+PMA-stimulated LTB4 synthesis. AACOCF3, a cPLA2 inhibitor, had no effect on either Cal or Cal+PMA-stimulated PAF synthesis. It reduced LTB4 synthesis, but it did so less effectively than Ro 25-4331. CoA-independent transacylase (CoAI-TA) activity in the AMs increased after stimulation and exposure to Ro 25-4331. SK&F 45905, a CoAI-TA inhibitor, reduced stimulated PAF synthesis by 30% to 40%. Patients with asthma had similar results except that cPLA2 had a greater role in stimulated LTB4 synthesis. These data indicate that PLA2 plays a direct role in human AM LT synthesis; both the cytosolic and secretory forms contribute to LT synthesis; PLA2 appears to have a more limited role in PAF synthesis, although it mediates the synergistic effect of PMA, probably via sPLA2; and CoAI-TA contributes to PAF synthesis during PLA2 inhibition. With the exception of the greater role for cPLA2 in stimulated LTB4 synthesis in the patients with asthma, the contributions of PLA2 and CoAI-TA to AM LT and PAF synthesis appear to be similar in normal subjects and patients with asthma.
Asunto(s)
Leucotrienos/biosíntesis , Macrófagos Alveolares/metabolismo , Factor de Activación Plaquetaria/biosíntesis , Acetiltransferasas/metabolismo , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Ácidos Araquidónicos/farmacología , Asma/metabolismo , Bencenosulfonatos/farmacología , Líquido del Lavado Bronquioalveolar , Calcimicina/farmacología , Calcio/fisiología , Células Cultivadas , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Humanos , Ionóforos/farmacología , Leucotrieno B4/biosíntesis , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Acetato de Tetradecanoilforbol/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
beta-ketoacyl-ACP synthetase III (KAS III) has been purified from avocado using a six-step purification procedure. The enzyme, which is cerulenin-insensitive and thiolactomycin-sensitive, was assayed using a partial component reaction: acetyl CoA:ACP transacylase (ACAT) activity. KAS III activity is distinguished from ACAT activity on the basis that the former is highly stimulated by the addition of malonyl CoA in the presence of malonyl-CoA:ACP transacylase, and the latter is not. KAS III and ACAT activity have been separated from each other thus providing the first evidence that these two discrete activities exist in higher plants. Both of these enzymes have been implicated in the initial reactions of fatty acid synthesis. KAS III was purified 134-fold using a combination of PEG precipitation, Fast Q, ammonium sulphate precipitation, Phenyl Sepharose and ACP-affinity chromatography. The enzyme requires Triton X-100 for solubility and is highly salt sensitive. The subunit molecular mass of 37 kDa has been identified by SDS-PAGE. The results of gel filtration analysis are consistent with the native enzyme being homodimeric. The native molecular mass of KAS III is 69 kDa and that of ACAT 18.5 kDa. The enzyme has a pH optimum of 7.0-7.5, which is similar to the pH optimum of the ACAT reaction. The Km for acetyl CoA is 12.5 microM and the Km for malonyl-ACP is 14 microM. Both KAS III and ACAT are sensitive to thiolactomycin inhibition. The results are discussed with respect to the potential role of acetyl CoA:ACP transacylase in plants.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/aislamiento & purificación , Acetiltransferasas/aislamiento & purificación , Frutas/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acetilcoenzima A/metabolismo , Acetiltransferasas/química , Acetiltransferasas/metabolismo , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Tampones (Química) , Cerulenina/farmacología , Concentración de Iones de Hidrógeno , Cinética , Malonil Coenzima A/metabolismo , Peso Molecular , Tiofenos/farmacologíaRESUMEN
Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants. The organism in unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP). The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants. The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I. The effects of inhibitors on the three different enzymes were noted. All carbon-carbon bond formation was inhibited by cerulenin. Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents. Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA. There was no evidence for a beta-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria. The roles of the acetyl-CoA-ACP transacylase and beta-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena. Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights.
Asunto(s)
Euglena gracilis/enzimología , Ácido Graso Sintasas/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Acetiltransferasas/metabolismo , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo , Aciltransferasas/metabolismo , Animales , Ácido Graso Sintasas/antagonistas & inhibidores , Ácidos Grasos/biosíntesis , Isoenzimas/metabolismoRESUMEN
Medium-chain fatty acids (FAs), found in storage lipids of certain plants, are an important renewable resource. Seeds of undomesticated California bay accumulate laurate (12:0), and a 12:0-acyl-carrier protein thioesterase (BTE) has been purified from this tissue. Sequencing of BTE enabled the cloning of a complementary DNA coding for a plastid-targeted preprotein. Expression of the complementary DNA in the seeds of Arabidopsis thaliana resulted in BTE activity, and medium chains accumulated at the expense of long-chain (greater than or equal to 16) FAs. Laurate became the most abundant FA species and was deposited in the storage triacylglycerols. These results demonstrate a mechanism for medium-chain FA synthesis in plants.
Asunto(s)
Acetiltransferasas/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Láuricos/metabolismo , Plantas/metabolismo , Acetiltransferasas/genética , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Secuencia de Aminoácidos , ADN/genética , Ácidos Grasos/aislamiento & purificación , Ingeniería Genética , Datos de Secuencia Molecular , Plantas/genética , Plantas Modificadas Genéticamente , Plásmidos , Semillas/metabolismoRESUMEN
The acyl-carrier protein (ACP) in Neurospora crassa mitochondria [Brody, S. & Mikolajczyk, S. (1988) Eur. J. Biochem. 173, 353-359] mediated a cerulenin-sensitive, de novo fatty acid synthesis independent of the fatty acid synthetase complex present in the cytoplasm. Incubation of mitochondria with [2-14C]malonate labeled only the ACP as indicated by autoradiography after SDS/PAGE. Under these in vitro conditions ATP was required for the initial acyl-ACP formation, but further elongation required either magnesium or the direct addition of NADPH. Labeled hexanoic (6:0) and caprylic (8:0) acids were detected as intermediates in the pathway, as well as hydroxymyristic acid. All of the intermediates, and the eventual product of the reaction, myristic acid (14:0), were released from the ACP by alkaline treatment. Pulse-chase experiments demonstrated the incorporation on to, and release of label from, the ACP. In vivo labeling of ACP with [2-14C]malonate was also detected and the label was in the form of hydroxymyristic acid. This newly discovered pathway is discussed from the standpoint of its possible role in providing acyl chains for mitochondrial lipids.
Asunto(s)
Acetiltransferasas/metabolismo , Ácidos Grasos/biosíntesis , Malonatos/metabolismo , Mitocondrias/metabolismo , Neurospora crassa/metabolismo , Neurospora/metabolismo , Acetiltransferasas/análisis , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Adenosina Trifosfato/farmacología , Autorradiografía , Ácidos Grasos/análisis , Magnesio/farmacología , Malonatos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Neurospora crassa/efectos de los fármacos , Neurospora crassa/enzimologíaRESUMEN
A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.
Asunto(s)
Acetiltransferasas/aislamiento & purificación , Escherichia coli/enzimología , Acetiltransferasas/antagonistas & inhibidores , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Etilmaleimida/farmacología , Yodoacetamida/farmacología , Cinética , Metilmetanosulfonato/farmacología , Peso MolecularRESUMEN
Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids. A trace amount of 13-cyclohexyltridecanoic acid is also present. Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids. The fatty acid synthetase system of this bacterium was studied. Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed. Sodium [14C]acetate and [14C]glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant. [14C]Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: [14C]leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and [14C]valine into 14-methylpentadecanoic acid. [14C]-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids. The fatty acid synthetase activity of the crude enzyme preparation of C. pusillum was reconstituted on the addition of acyl carrier protein. This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer. The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids. The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.
Asunto(s)
Bacterias/enzimología , Ácido Graso Sintasas/análisis , Acetiltransferasas/metabolismo , Acilcoenzima A/metabolismo , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Cromatografía de Gases , Escherichia coli/enzimología , Ácidos Grasos/metabolismo , NADP/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
Thiolactomycin, an antibiotic with the structure of (4S)-(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-++ +thiolide, selectively inhibits type II fatty acid synthases. The mode of the thiolactomycin action on the fatty acid synthase system of Escherichia coli was investigated. Of the six individual enzymes of the fatty acid synthase system, [acyl-carrier-protein] (ACP) acetyltransferase and 3-oxoacyl-ACP synthase were inhibited by thiolactomycin. On the other hand, the other enzymes were not affected by this antibiotic. The thiolactomycin inhibition of the fatty acid synthase system was reversible. As to ACP acetyltransferase, the inhibition was competitive with respect to ACP and uncompetitive with respect to acetyl-CoA. As to 3-oxoacyl-ACP synthase, the inhibition was competitive with respect to malonyl-ACP and noncompetitive with respect to acetyl-ACP. The thiolactomycin action on the fatty acid synthase system was compared with that of cerulenin.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/enzimología , Ácido Graso Sintasas/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Acetiltransferasas/antagonistas & inhibidores , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Cerulenina/farmacología , Escherichia coli/efectos de los fármacos , Lactonas/farmacología , TiofenosAsunto(s)
Ácido Graso Sintasas/análisis , Plantas/enzimología , Acetiltransferasas/aislamiento & purificación , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Animales , Cloroplastos/enzimología , Escherichia coli/enzimología , Ácido Graso Sintasas/biosíntesis , Ácidos Palmíticos/biosíntesis , Ratas , Ácidos Esteáricos/biosíntesisRESUMEN
When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.
Asunto(s)
Acetiltransferasas/aislamiento & purificación , Arsenitos , Plantas/enzimología , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Arsénico/farmacología , Ácido Graso Sintasas/aislamiento & purificación , Ácido Graso Sintasas/metabolismo , Cinética , Especificidad de la Especie , Especificidad por SustratoAsunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Acetiltransferasas/antagonistas & inhibidores , Aciltransferasas/antagonistas & inhibidores , Coenzima A/análogos & derivados , Ácido Graso Sintasas/antagonistas & inhibidores , Hígado/enzimología , Acetilcoenzima A/farmacología , S-Acetiltransferasa de la Proteína Transportadora de Grupos Acilo , Aminoácidos/farmacología , Animales , Coenzima A/metabolismo , Coenzima A/farmacología , Columbidae , Cisteína/farmacología , Activación Enzimática/efectos de los fármacos , Yodoacetamida/farmacología , Cinética , Malonil Coenzima A/farmacología , Panteteína/farmacología , EspectrofotometríaRESUMEN
We have analyzed a mutation of Bacillus subtilis (bfmB) that results in an acyl-CoA:acyl-carrier-protein transacylase with low affinity for branched acyl-CoA substrates; it maps in the acf-hisH region of the chromosome. The aceA mutation, present in the parent of the bfmB mutant, causes a deficiency in pyruvate dehydrogenase and maps in the pycA-pyrA region. Strains carrying the bfmB mutation synthesize branched-chain fatty acids at a rate sufficient for normal growth only if branched acyl-CoA precursors are present in the medium. They grow well if the medium is supplemented with 0.1 mM 2-methylbutyrate, isobutyrate or isovalerate, or with 1.0 mM isoleucine or valine; leucine does not support growth. Growth supported by valine and isoleucine is inhibited by butyrate and other straight short-chain fatty acids at concentrations (0.1 mM) which do not inhibit growth of the standard strain; the inhibition is prevented by short branched fatty acids which are converted to long-chain fatty acids appearing as activity of B. subtilis is controlled by separate enzymatic sites for the acyl-CoA precursors of branched and straight-chain fatty acids. Whether these sites are contained in one or two enzymes is not known.