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1.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 6): 758-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26057808

RESUMO

The gene product of A1887 from Ralstonia eutropha (ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of ß-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purified ReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, with Km, Vmax and kcat values of 158 µM, 32 mM min(-1) and 5 × 10(6) s(-1), respectively. The ReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 M HEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 129.52, c = 114.13 Å, α = ß = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.


Assuntos
Acetil-CoA C-Aciltransferase/química , Acil Coenzima A/química , Proteínas de Bactérias/química , Cupriavidus necator/enzimologia , Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Cupriavidus necator/química , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
2.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 3): 316-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24598917

RESUMO

Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. ß-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Šon a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54 Å(3) Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.


Assuntos
Acetil-CoA C-Aciltransferase/química , Proteínas de Bactérias/química , Cupriavidus necator/enzimologia , Acetil-CoA C-Aciltransferase/biossíntese , Acetil-CoA C-Aciltransferase/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli
3.
FEBS J ; 273(11): 2374-87, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16704412

RESUMO

A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.


Assuntos
Acetil-CoA C-Aciltransferase/metabolismo , Alcaligenes faecalis/enzimologia , Acetil-CoA C-Aciltransferase/química , Acetil-CoA C-Aciltransferase/isolamento & purificação , Alcaligenes faecalis/ultraestrutura , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Cinética , Espectrometria de Massas , Microscopia Eletrônica , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Especificidade por Substrato
4.
Mol Cell Biochem ; 269(1-2): 27-36, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15786714

RESUMO

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a beta-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 degrees C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. beta-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 microM and 25 microM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of beta-ketothiolase.


Assuntos
Acetil-CoA C-Aciltransferase/química , Acetil-CoA C-Aciltransferase/isolamento & purificação , Thermus thermophilus/enzimologia , Acetil-CoA C-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Temperatura Alta , Poliésteres/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação
5.
Protein Expr Purif ; 35(2): 320-6, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15135409

RESUMO

Mitochondrial 3-ketoacyl-CoA thiolase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned a cDNA of rat mitochondrial 3-ketoacyl-CoA thiolase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. The cloned cDNA was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 92% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial 3-ketoacyl-CoA thiolase was 25U/mg. It has been proposed that His352 is a catalytic residue responsible for activation of coenzyme A by deprotonation of a sulfhydryl group. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that His352 is a catalytic residue of rat mitochondrial 3-ketoacyl-CoA thiolase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial 3-ketoacyl-CoA thiolase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by 3-ketoacyl-CoA thiolase.


Assuntos
Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/isolamento & purificação , Histidina/química , Mitocôndrias/enzimologia , Acetil-CoA C-Aciltransferase/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , Primers do DNA , DNA Complementar , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos , Ratos , Homologia de Sequência de Aminoácidos
6.
Protein Expr Purif ; 33(1): 25-33, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14680958

RESUMO

The glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5'- and 3'-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.


Assuntos
Acetil-CoA C-Aciltransferase/biossíntese , Acetil-CoA C-Aciltransferase/genética , Glioxissomos/enzimologia , Helianthus/enzimologia , Acetil-CoA C-Aciltransferase/isolamento & purificação , Acetil-CoA C-Aciltransferase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cotilédone/enzimologia , Cotilédone/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/genética , Helianthus/genética , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
7.
Circ Res ; 93(3): e26-32, 2003 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-12869391

RESUMO

Trimetazidine acts as an effective antianginal clinical agent by modulating cardiac energy metabolism. Recent published data support the hypothesis that trimetazidine selectively inhibits long-chain 3-ketoacyl CoA thiolase (LC 3-KAT), thereby reducing fatty acid oxidation resulting in clinical benefit. The aim of this study was to assess whether trimetazidine and ranolazine, which may also act as a metabolic modulator, are specific inhibitors of LC 3-KAT. We have demonstrated that trimetazidine and ranolazine do not inhibit crude and purified rat heart or recombinant human LC 3-KAT by methods that both assess the ability of LC 3-KAT to turnover specific substrate, and LC 3-KAT activity as a functional component of intact cellular beta-oxidation. Furthermore, we have demonstrated that trimetazidine does not inhibit any component of beta-oxidation in an isolated human cardiomyocyte cell line. Ranolazine, however, did demonstrate a partial inhibition of beta-oxidation in a dose-dependent manner (12% at 100 micromol/L and 30% at 300 micromol/L). Both trimetazidine (10 micromol/L) and ranolazine (20 micromol/L) improved the recovery of cardiac function after a period of no flow ischemia in the isolated working rat heart perfused with a buffer containing a relatively high concentration (1.2 mmol/L) of free fatty acid. In summary, both trimetazidine and ranolazine were able to improve ischemic cardiac function but inhibition of LC 3-KAT is not part of their mechanism of action. The full text of this article is available online at http://www.circresaha.org.


Assuntos
Acetil-CoA C-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mitocôndrias Cardíacas/enzimologia , Trimetazidina/farmacologia , Vasodilatadores/farmacologia , Acetanilidas , Acetil-CoA C-Aciltransferase/isolamento & purificação , Acetil-CoA C-Aciltransferase/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Técnicas In Vitro , Masculino , Mitocôndrias Cardíacas/química , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/fisiopatologia , Miocárdio/enzimologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Oxirredução/efeitos dos fármacos , Piperazinas/farmacologia , Ranolazina , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
8.
Planta ; 214(4): 597-607, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11925043

RESUMO

Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).


Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Cotilédone/enzimologia , Glioxissomos/enzimologia , Helianthus/enzimologia , Acetil-CoA C-Acetiltransferase/isolamento & purificação , Acetil-CoA C-Aciltransferase/isolamento & purificação , Acil Coenzima A/metabolismo , Ácidos Graxos/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Palmitoil Coenzima A/metabolismo
9.
J Bacteriol ; 184(1): 207-15, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11741862

RESUMO

The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 +/- 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 +/- 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.


Assuntos
Acetil-CoA C-Aciltransferase/metabolismo , Adipatos/metabolismo , Benzoatos/metabolismo , Clorobenzenos/metabolismo , Coenzima A-Transferases/metabolismo , Pseudomonas/enzimologia , Acetil-CoA C-Aciltransferase/isolamento & purificação , Sequência de Aminoácidos , Biodegradação Ambiental , Coenzima A-Transferases/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de Proteína , Especificidade por Substrato
10.
Planta Med ; 67(7): 659-64, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11582546

RESUMO

Nine lanostane and serratane-type triterpenes including two unknown compounds were isolated from the stem bark of Pinus luchuensis. These new compounds were characterized as 3-oxolanost-9(11)-ene-24S,25-diol (1) and 29-acetoxy-3beta-methoxyserrat-14-en-21alpha-ol (2) on the basis of spectroscopic evidence. Some of these triterpenes were tested for the inhibitory effect on DNA topoisomerase II activity. Compound 1 showed a slightly less potent inhibitory activity with an IC(50) value of 186 microM.


Assuntos
Acetil-CoA C-Aciltransferase/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Pinus/química , Casca de Planta/química , Extratos Vegetais/farmacologia , Inibidores da Topoisomerase II , Triterpenos/isolamento & purificação , Acetil-CoA C-Aciltransferase/química , Acetil-CoA C-Aciltransferase/farmacologia , Etoposídeo/farmacologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Triterpenos/química , Triterpenos/farmacologia
11.
Biochem J ; 341 ( Pt 3): 713-23, 1999 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10417336

RESUMO

The fibroblast growth factor (FGF) family is composed of polypeptides with sequence identity which signal through transmembrane tyrosine kinase receptors. We report here the purification from bovine brain microsomes of an FGF-2-binding complex composed of three proteins of apparent molecular masses 150 kDa, 79 kDa and 46 kDa. Only the 150 kDa and 79 kDa proteins bound FGF-2 in cross-linking and ligand-blotting experiments. Binding of FGF-2 to p79 is enhanced in the presence of calcium. Peptide sequences allowed the identification of p150 and the cloning of the cDNAs encoding p79 and p46. The deduced amino acid sequence of p79 reveals high similarity to those of gastrin-binding protein and mitochondrial enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase. p46 is similar to mitochondrial ketoacyl-CoA thiolase. Stable transfection of FR3T3 rat fibroblast cells with p79 cDNA analysed by electron microscopy following immunolabelling of ultra-thin cryosections revealed a localization of p79 in the secretory pathway, mainly in the endoplasmic reticulum and the Golgi region, where it is specifically associated with the molecular chaperone calnexin. In vivo a protein similar to the Golgi protein MG-160 forms a complex with FGF-2 and p79.


Assuntos
Acetil-CoA C-Aciltransferase/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Bovinos , Clonagem Molecular , DNA Complementar , Microssomos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Ratos , Homologia de Sequência de Aminoácidos
12.
Biochim Biophys Acta ; 1437(2): 136-41, 1999 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-10064897

RESUMO

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.


Assuntos
Acetil-CoA C-Aciltransferase/biossíntese , Fígado/enzimologia , Acetil-CoA C-Aciltransferase/isolamento & purificação , Sequência de Aminoácidos , Animais , Clofibrato , Indução Enzimática , Estabilidade Enzimática , Isoenzimas/biossíntese , Masculino , Microcorpos/enzimologia , Dados de Sequência Molecular , Ratos , Ratos Wistar , Especificidade por Substrato
13.
J Bacteriol ; 180(8): 1979-87, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9555876

RESUMO

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-beta-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form beta-ketovaleryl-CoA. This activity has generally been attributed to the beta-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a beta-ketothiolase from R. eutropha that is capable of forming beta-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of beta-hydroxyvalerate derived from propionyl-CoA. We also report an additional beta-ketothiolase, designated BktC, that probably serves as a secondary route toward beta-hydroxyvalerate production.


Assuntos
Acetil-CoA C-Aciltransferase/metabolismo , Alcaligenes/enzimologia , Polímeros/metabolismo , Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/isolamento & purificação , Alcaligenes/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Bacterianos , Teste de Complementação Genética , Genótipo , Cinética , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
Electrophoresis ; 17(10): 1643-50, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8957197

RESUMO

Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.


Assuntos
Eletroforese em Gel Bidimensional , Espectrometria de Massas , Miocárdio/química , Proteínas/isolamento & purificação , Acetil-CoA C-Aciltransferase/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Creatina Quinase/isolamento & purificação , Humanos , Isocitrato Desidrogenase/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Mioglobina/isolamento & purificação , Mapeamento de Peptídeos , Tripsina
15.
Structure ; 2(9): 797-808, 1994 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-7812714

RESUMO

BACKGROUND: The peroxisomal enzyme 3-ketoacyl-coenzyme A thiolase of the yeast Saccharomyces cerevisiae is a homodimer with 417 residues per subunit. It is synthesized in the cytosol and subsequently imported into the peroxisome where it catalyzes the last step of the beta-oxidation pathway. We have determined the structure of this thiolase in order to study the reaction mechanism, quaternary associations and intracellular targeting of thiolases generally, and to understand the structural basis of genetic disorders associated with human thiolases. RESULTS: Here we report the crystal structure of unliganded yeast thiolase refined at 2.8 A resolution. The enzyme comprises three domains; two compact core domains having the same fold and a loop domain. Each of the two core domains is folded into a mixed five-stranded beta-sheet covered on each side by helices and the two are assembled into a five-layered alpha beta alpha beta alpha structure. The central layer is formed by two helices, which point with their amino termini towards the active site. The loop domain, which is to some extent stabilized by interactions with the other subunit, runs over the surface of the two core domains, encircling the active site of its own subunit. CONCLUSIONS: The crystal structure of thiolase shows that the active site is a shallow pocket, shaped by highly conserved residues. Two conserved cysteines and a histidine at the floor of this pocket probably play key roles in the reaction mechanism. The two active sites are on the same face of the dimer, far from the amino and carboxyl termini of both subunits and the disordered amino-terminal import signal sequence.


Assuntos
Acetil-CoA C-Aciltransferase/química , Microcorpos/enzimologia , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/enzimologia , Acetil-CoA C-Aciltransferase/isolamento & purificação , Sequência de Aminoácidos , Clonagem Molecular , Gráficos por Computador , Sequência Conservada , Cristalografia por Raios X/métodos , Cisteína , Histidina , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
16.
Yeast ; 10(9): 1173-82, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7754706

RESUMO

A molecular understanding of peroxisome biogenesis depends upon the analysis of peroxisomal proteins. Here we describe the isolation of the 3-oxoacyl-CoA thiolase of the peroxisomal beta-oxidation system from Saccharomyces cerevisiae as a dimer of identical subunits, each with a molecular mass of 45 kDa. Monospecific polyclonal antibodies were raised against the purified enzyme, and its peroxisomal origin was demonstrated by immunoblotting of subcellular fractions as well as by immunogold labelling. We also show that these antibodies could be suitable for an immunofluorescence microscopy screening of yeast mutants affected in peroxisome assembly.


Assuntos
Acetil-CoA C-Aciltransferase/imunologia , Acetil-CoA C-Aciltransferase/isolamento & purificação , Microcorpos/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/ultraestrutura , Acetil-CoA C-Aciltransferase/análise , Animais , Anticorpos/análise , Masculino , Microcorpos/genética , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Coelhos , Saccharomyces cerevisiae/genética
17.
Eur J Biochem ; 216(2): 607-13, 1993 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-7916689

RESUMO

Using a Yarrowia lipolytica genomic library, several overlapping clones of the peroxisomal 3-oxoacyl-CoA-thiolase gene, POT1, were isolated. The library was prepared in the bacterial expression vector lambda gt11, thus allowing an immunological screening of recombinant bacteriophages with specific antibodies raised against purified peroxisomal thiolase. The isolated POT1 clones hybridized to a 1.4 kb RNA species, which was induced approximately 30-fold when oleate was the carbon source. A 3634-bp segment of the cloned DNA was sequenced. This segment contained, on both strands, three major overlapping open-reading frames of 678, 1122 and 1242 bp. Northern-hybridization analysis showed that only the largest of these reading frames was transcribed. It encodes a protein of 414 amino acids and molecular mass 43.059 kDa. Its deduced amino acid sequence has 30-60% identity and 50-70% sequence similarity when compared to other known thiolases. According to both the amount (68-71%) and location of conserved amino acids, the encoded protein belongs to the peroxisomal rather than the mitochondrial or cytoplasmic class of thiolases. Compared to bacterial and yeast cytosolic thiolases, the POT1 gene product contains a N-terminal extension of 25 amino acids which clearly differs from typical mitochondrial import signals. One of the isolated clones contained, in addition to the POT1 coding sequence, 784 bp of the corresponding 5' flanking region. Nevertheless, it was efficiently expressed in Escherichia coli suggesting the correct recognition of this fungal promoter by the prokaryotic transcriptional and translational machinery. The Y. lipolytica genomic POT1 gene was disrupted by replacing 120 bp of its coding sequence with 2.7 kbp of DNA including the Y. lipolytica LEU2 gene. The resulting delta pot1::LEU2 cells were free of immunologically cross-reacting thiolase. Western-blot analysis showed that the product of the non-disrupted gene had a molecular mass of approximately 42 kDa. This corresponds well to the molecular mass of purified Y. lipolytica peroxisomal thiolase. Disruption of POT1 abolished the ability of Y. lipolytica cells to grow on solid media with oleate as a carbon source. This inability to grow in the presence of oleate suggests both the catabolic function of POT1 and the absence of additional catabolic thiolases in Y. lipolytica. However, the delta pot1::LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher-chain-length homologues. Hence, another, POT1-independent and biosynthetic 3-oxoacyl-CoA thiolase must be responsible for this reaction in Y. lipolytica.


Assuntos
Acetil-CoA C-Aciltransferase/genética , Regulação da Expressão Gênica , Microcorpos/enzimologia , Saccharomycetales/enzimologia , Acetil-CoA C-Aciltransferase/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Dados de Sequência Molecular , Saccharomycetales/genética
19.
J Biochem ; 112(6): 845-8, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1363552

RESUMO

The presence of two types of thiolases, acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase, was demonstrated in peroxisomes of n-alkane-grown Candida tropicalis [Kurihara, T., Ueda, M., & Tanaka, A. (1989) J. Biochem. 106, 474-478], while acetoacetyl-CoA thiolase was also shown to be present in cytosol. The activity of the enzyme in cytosol was constant irrespective of culture conditions, while the peroxisomal enzyme was inducibly synthesized in the alkane-grown yeast cells. These results indicate that peroxisomal acetoacetyl-CoA thiolase participates in alkane degradation, while the cytosolic enzyme is associated with other fundamental metabolic processes, probably sterol biosynthesis, because this enzyme can catalyze the first step of the sterol biosynthesis. 3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, a key regulatory enzyme of sterol biosynthesis, was found to be localized exclusively in microsomes of the alkane-grown yeast cells. These results suggest that yeast peroxisomes do not contribute to sterol biosynthesis, unlike the case of mammalian cells.


Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Alcanos/metabolismo , Candida/enzimologia , Esteróis/biossíntese , Acetil-CoA C-Acetiltransferase/isolamento & purificação , Acetil-CoA C-Aciltransferase/isolamento & purificação , Candida/crescimento & desenvolvimento , Candida/metabolismo , Fracionamento Celular/métodos , Citosol/enzimologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Cinética , Microcorpos/enzimologia , Modelos Biológicos , Frações Subcelulares/enzimologia
20.
J Biol Chem ; 267(2): 1034-41, 1992 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-1730633

RESUMO

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/isolamento & purificação , Acetil-CoA C-Aciltransferase/isolamento & purificação , Enoil-CoA Hidratase/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Complexos Multienzimáticos/isolamento & purificação , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Animais , Western Blotting , Cromatografia Líquida , Detergentes , Eletroforese em Gel de Poliacrilamida , Enoil-CoA Hidratase/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Peso Molecular , Complexos Multienzimáticos/metabolismo , Oxirredução , Mapeamento de Peptídeos , Ratos , Ratos Endogâmicos , Especificidade por Substrato
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