RESUMEN
Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Aciltransferasas/metabolismo , Coenzima A Transferasas/metabolismo , Peptococcaceae/enzimología , Polihidroxialcanoatos/biosíntesis , Thermus thermophilus/enzimología , Ácido 3-Hidroxibutírico/metabolismo , Acetato CoA Ligasa/aislamiento & purificación , Acetilcoenzima A/metabolismo , Aciltransferasas/aislamiento & purificación , Coenzima A Transferasas/aislamiento & purificación , Estabilidad de Enzimas , Hidroxibutiratos/metabolismo , Ácido Láctico/metabolismo , Peptococcaceae/metabolismo , Poliésteres/metabolismo , Temperatura , Thermus thermophilus/metabolismoRESUMEN
The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PP(i)) and was only moderately inhibited by PP(i). The breakdown of PP(i) was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PP(i) (K(M) = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PP(i). However, it cannot be excluded that other PP(i)-dependent enzymes take advantage of the remaining PP(i) and contribute to the energy balance of the cell.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Proteínas Arqueales/metabolismo , Methanosarcinales/enzimología , Pirofosfatasas/metabolismo , Acetato CoA Ligasa/genética , Acetato CoA Ligasa/aislamiento & purificación , Acetilcoenzima A/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Arqueales/genética , Clonación Molecular , Difosfatos/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica Arqueal , Regulación Enzimológica de la Expresión Génica , Genes Arqueales , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Methanosarcinales/genética , Conformación Molecular , Pirofosfatasas/genética , Pirofosfatasas/aislamiento & purificación , SolubilidadRESUMEN
Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of â¼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Adenosina Monofosfato/metabolismo , Desulfurococcaceae/enzimología , Desulfurococcaceae/metabolismo , Proteínas de la Membrana/metabolismo , Acetato CoA Ligasa/química , Acetato CoA Ligasa/aislamiento & purificación , Proteínas Arqueales/química , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Electroforesis en Gel Bidimensional , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Microscopía , Modelos Biológicos , Peso Molecular , Multimerización de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Hidrolasas/aislamiento & purificación , Hidrolasas/metabolismo , Mycobacterium tuberculosis/enzimología , Acetato CoA Ligasa/genética , Acetatos/química , Acetilación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Hidrolasas/genética , Datos de Secuencia Molecular , Mutación/genética , Sirtuinas/genética , Sirtuinas/metabolismo , Especificidad por SustratoRESUMEN
A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a methyl group, coenzyme A, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermolecular CO transport. Here, we report a 2.5 A resolution structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calculation program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid composition surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.
Asunto(s)
Acetato CoA Ligasa/química , Aldehído Oxidorreductasas/química , Complejos Multienzimáticos/química , Xenón/química , Acetato CoA Ligasa/aislamiento & purificación , Aldehído Oxidorreductasas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Bacilos Grampositivos Asporogénicos Irregulares , Modelos Químicos , Modelos Moleculares , Complejos Multienzimáticos/aislamiento & purificación , Valor Predictivo de las Pruebas , Unión ProteicaRESUMEN
The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140 degrees rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.
Asunto(s)
Acetato CoA Ligasa/química , Acetato CoA Ligasa/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Salmonella enterica/enzimología , Acetato CoA Ligasa/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Catálisis , Cristalización/métodos , Cristalografía por Rayos X , Glicina/química , Glicina/genética , Cinética , Lisina/química , Lisina/genética , Conformación Molecular , Estructura Molecular , Proteínas Mutantes/aislamiento & purificación , Salmonella enterica/genética , Especificidad por Sustrato/genética , Valina/química , Valina/genéticaRESUMEN
AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Methanobacteriaceae/enzimología , Acetato CoA Ligasa/química , Acetato CoA Ligasa/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Coenzima A/metabolismo , Isoleucina/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Saccharomyces cerevisiae/enzimología , Salmonella enterica/enzimología , Alineación de Secuencia , Especificidad por Sustrato , Treonina/metabolismo , Triptófano/metabolismo , Valina/metabolismoRESUMEN
Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.
Asunto(s)
Acetato CoA Ligasa/genética , Acetato CoA Ligasa/aislamiento & purificación , Adenosina Monofosfato/metabolismo , Expresión Génica/genética , Haloarcula marismortui/enzimología , Filogenia , Acetato CoA Ligasa/química , Acetato CoA Ligasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Estabilidad de Enzimas , Haloarcula marismortui/efectos de los fármacos , Haloarcula marismortui/genética , Humanos , Datos de Secuencia Molecular , Peso Molecular , Cloruro de Potasio/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Especificidad por SustratoRESUMEN
Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.
Asunto(s)
Acetato CoA Ligasa/química , Adenosina Monofosfato/química , Coenzima A/química , Acetato CoA Ligasa/aislamiento & purificación , Acetilación , Acilcoenzima A/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Unión Competitiva , Coenzima A Ligasas/antagonistas & inhibidores , Coenzima A Ligasas/química , Cristalización , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Datos de Secuencia Molecular , Conformación Proteica , Salmonella enterica/enzimologíaRESUMEN
Acetyl-CoA synthetase (AceCS), which catalyzes the activation of acetate to produce acetyl-CoA, was found to have a much greater Km value for acetate in liver mitochondria than that in the heart mitochondria of rats, indicating that two different types of AceCS are located in the liver and heart mitochodria. Recently, Fujino et al. reported that mouse heart mitochondrial AceCS, designated AceCS2, was expressed in a wide range of tissues, however, it was apparently absent from the liver. In this study, liver mitochondrial AceCS activity, but not heart AceCS2, was greatly induced in di(2-ethylhexyl)phthalate (DEHP)-treated rats. We purified and characterized the rat liver mitochondrial AceCS. The molecular mass of the enzyme estimated by SDS-PAGE was -58 kDa, which was quite different from that of the heart mitochondrial enzyme, AceCS2. The calculated Km value for the acetate of the partially purified liver enzyme was much greater, being about 100 times that of heart enzyme, AceCS2.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Mitocondrias Hepáticas/enzimología , Acetato CoA Ligasa/efectos de los fármacos , Animales , Dietilhexil Ftalato/administración & dosificación , Electroforesis en Gel de Poliacrilamida , Masculino , Mitocondrias Cardíacas/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
The ATP-dependent activation of short- and medium-chain fatty acids to their respective CoA thioester adducts was investigated in the colonic mucosa from swine. Subcellular fractionation of a homogenate of the mucosa from the entire length of the colon revealed a predominantly mitochondrial localization for activity toward fatty acids ranging from propionate through laurate. These activities could be released from mitochondria in soluble form by freeze-thaw lysis. Purification of these activities revealed that they all appeared to reside with a single enzyme. This suggests that the entire colon contains a single form of medium-chain fatty acid:CoA ligase (MCFA:CoA ligase). The ligase also had activity toward benzoate and salicylate, although this activity was significantly lower than activity toward medium-chain fatty acids. The enzyme had the highest activity at Vmax with butyrate as substrate and had the lowest Km for octanoate. Butyrate and octanoate were mutually inhibitory. Activity toward both substrates was also efficiently inhibited by cyclohexane carboxylate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be ca. 46,500. These data indicate that the colonic MCFA:CoA ligase is significantly different from the hepatic and kidney MCFA:CoA ligases.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Acetato CoA Ligasa/fisiología , Colon/metabolismo , Ácidos Grasos Volátiles/metabolismo , Mucosa Intestinal/metabolismo , Acetato CoA Ligasa/química , Animales , Fraccionamiento Celular , Cromatografía en Gel , Mitocondrias , Peso Molecular , Ratas , Especificidad por Sustrato , PorcinosAsunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Acetato CoA Ligasa/metabolismo , Pyrococcus furiosus/enzimología , Acetato CoA Ligasa/análisis , Cromatografía/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Durapatita , Indicadores y Reactivos , Isoenzimas/análisis , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Pyrococcus furiosus/crecimiento & desarrolloRESUMEN
Giardia lamblia, an amitochondriate eukaryote, contains acetyl-CoA synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350-fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed acetyl-CoA synthetase (AMP-forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Acetato CoA Ligasa/metabolismo , Adenosina Difosfato/biosíntesis , Giardia lamblia/enzimología , Acetato CoA Ligasa/química , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Adenosina Difosfato/metabolismo , Animales , Coenzima A/metabolismo , Giardia lamblia/metabolismo , Cinética , Magnesio/metabolismo , Fosfatos/metabolismo , Especificidad por SustratoRESUMEN
Acetyl-CoA synthetase from bovine heart has been purified to homogeneity and been crystallized. The purification procedure involves ammonium sulfate precipitation and subsequent column chromatography on DEAE-Sepharose, Blue-Sepharose, CoA-Agarose and Superose 6. The purified enzyme has a specific activity of 45 units/mg protein, and its molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis is approximately 72,000. The purified enzyme specifically utilizes acetate, ATP and CoA. Apparent Km values of the purified enzyme for acetate, CoA, and ATP were 0.16 mM, 0.14 mM and 0.25 mM, respectively. Limited digestion with trypsin, subtilisin BPN' and chymotrypsin revealed that the enzyme contains a 56 k segment resistant to these proteases. Secondary structure contents of the purified enzyme and the 56 k tryptic fragment were analyzed by circular dichroism measurement. The intact molecule contains 30% alpha-helix and 30% beta-structure, and trypsin digests alpha-helix rich regions more substantially. Western blot analysis of rat tissue homogenates by specific antibodies against the purified enzyme indicated that the 72 k enzyme is present in a wide variety of tissues and is most abundant in heart and kidney.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Miocardio/enzimología , Estructura Secundaria de Proteína , Acetato CoA Ligasa/química , Secuencia de Aminoácidos , Animales , Bovinos , Dicroismo Circular , Clonación Molecular , Cristalografía por Rayos X , Hidrólisis , Cinética , Masculino , Datos de Secuencia Molecular , Ratas , Ratas WistarRESUMEN
Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Acetato CoA Ligasa/metabolismo , Penicilinas/biosíntesis , Penicillium chrysogenum/enzimología , Aspergillus nidulans/enzimología , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Cinética , Peso Molecular , Penicilinas/aislamiento & purificación , TermodinámicaRESUMEN
Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Coenzima A Ligasas/aislamiento & purificación , Rhizobiaceae/enzimología , Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Ácido Acético , Adenosina Trifosfato/metabolismo , Cationes Bivalentes , Cromatografía , Coenzima A/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Cloruro de Magnesio/farmacología , Peso Molecular , Nucleótidos/metabolismo , Especificidad por SustratoRESUMEN
In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Coenzima A Ligasas/aislamiento & purificación , Euryarchaeota/enzimología , Acetato CoA Ligasa/antagonistas & inhibidores , Acetatos/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/metabolismo , Difosfatos/farmacología , Cinética , Peso Molecular , Especificidad por SustratoAsunto(s)
Acetato CoA Ligasa/análisis , Coenzima A Ligasas/análisis , Acetato CoA Ligasa/antagonistas & inhibidores , Acetato CoA Ligasa/aislamiento & purificación , Animales , Citosol/enzimología , Concentración de Iones de Hidrógeno , Hígado/enzimología , Peso Molecular , Ratas , Especificidad por SustratoRESUMEN
Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%). The apparent Kms for acetate, coenzyme A, ATP and MgCl2 were determined and found to be 52.5 microM, 50.5 microM, 570 microM and 1.5 mM, respectively. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.
Asunto(s)
Acetato CoA Ligasa/aislamiento & purificación , Coenzima A Ligasas/aislamiento & purificación , Hígado/enzimología , Acetato CoA Ligasa/metabolismo , Animales , Citosol/enzimología , Femenino , Cinética , Peso Molecular , Ratas , Ratas Endogámicas , Especificidad por SustratoRESUMEN
Purification of components of heart mitochondria activating short chain fatty acids prepared from tissue of lactating Holstein cows demonstrated predominantly one acyl CoA synthetase, acetyl CoA synthetase activating acetate, and propionate. Activity of butyryl CoA synthetase was low. Propionyl CoA synthetase characteristically in bovine liver and kidney tissue could not be demonstrated in heart mitochondria. Thus, of the ruminally derived volatile fatty acids only acetate can be used by heart mitochondria as a primary energy source because of small quantities of propionate in peripheral blood. Acetyl CoA synthetase was a glycoprotein composed of a single polypeptide chain of apparent molecular weight 67,500. The Michaelis-Menten constant for acetate was 1.8 x 10(-4)M. By comparison with literature for blood acetate concentration we concluded that enzyme is saturated with substrate at all physiological concentrations of acetate. These kinetic properties ensure a constant supply of acetate as an energy source for maintaining heart function in ruminants.