RESUMO
Enoyl reductase (ENR), an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the front-line antituberculosis drug isoniazid. Analysis of the structures of complexes of Escherichia coli ENR with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2' hydroxyl of the nicotinamide ribose and a boron atom in the drugs to generate a tight, noncovalently bound bisubstrate analog. This analysis has implications for the structure-based design of inhibitors of ENR, and similarities to other oxidoreductases suggest that mimicking this molecular linkage may have generic applications in other areas of medicinal chemistry.
Assuntos
Antibacterianos/metabolismo , Compostos de Boro/metabolismo , Inibidores Enzimáticos/metabolismo , Ácido Graxo Sintases/química , NAD/metabolismo , Oxirredutases/química , Antibacterianos/farmacologia , Sítios de Ligação , Compostos de Boro/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Resistência Microbiana a Medicamentos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Ácido Graxo Sintase Tipo II , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. RESULTS: The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. CONCLUSIONS: A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Brassica/enzimologia , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase , Adenina/química , Adenina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
BACKGROUND: Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans-delta 2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADH-dependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADH-dependent enzyme are found during lipid deposition in maturing seeds of oilseed rape (Brassica napus). RESULTS: The crystal structure of an ENR-NAD binary complex has been determined at 1.9 A resolution and consists of a homotetramer in which each subunit forms a single domain comprising a seven-stranded parallel beta sheet flanked by seven alpha helices. The subunit has a topology highly reminiscent of a dinucleotide-binding fold. The active site has been located by difference Fourier analysis of data from crystals equilibrated in NADH. CONCLUSIONS: The structure of ENR shows a striking similarity with the epimerases and short-chain alcohol dehydrogenases, in particular, 3 alpha,20 beta-hydroxysteroid dehydrogenase (HSD). The similarity with HSD extends to the conservation of a catalytically important lysine that stabilizes the transition state and to the use of a tyrosine as a base--with subtle modifications arising from differing requirements of the reduction chemistry.
Assuntos
Brassica/enzimologia , Cristalografia por Raios X , Oxirredutases/química , Sequência de Aminoácidos , Anabaena/enzimologia , Sítios de Ligação , Sequência Conservada , Cortisona Redutase/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Escherichia coli/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , NAD/química , NAD/metabolismo , Nucleotídeos/metabolismo , Oxirredução , Conformação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.
Assuntos
Glicerol-3-Fosfato O-Aciltransferase/química , Sequência de Aminoácidos , Sítios de Ligação , Glicerofosfatos/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Especificidade por Substrato , Verduras/enzimologiaRESUMO
Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO2 fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O2 evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected. The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient DL-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity. It is concluded that ribose 5-phosphate inhibits photosynthetic O2 evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.
Assuntos
Cloroplastos/metabolismo , Oxigênio/metabolismo , Pentosefosfatos/farmacologia , Fotossíntese/efeitos dos fármacos , Ribose/farmacologia , Dióxido de Carbono/metabolismo , Cloroplastos/efeitos dos fármacos , Ditiotreitol/farmacologia , Glicerofosfatos/metabolismo , PlantasRESUMO
When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, 14CO2 fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O2 evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway.
Assuntos
Dióxido de Carbono/metabolismo , Cloroplastos/metabolismo , Comitês de Ética em Pesquisa , Frutosefosfatos/metabolismo , Hexosedifosfatos/metabolismo , Oxigênio/metabolismo , Fotossíntese , Ditiotreitol/farmacologia , Comitês de Ética em Pesquisa/normas , Frutose-Bifosfatase/metabolismo , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Consumo de Oxigênio , Fotossíntese/efeitos dos fármacos , PlantasRESUMO
The NADH-dependent enoyl-ACP reductase from oil seed rape (Brassica napus) was inactivated by treatment with phenylglyoxal, a reagent which specifically modifies arginine residues. The inhibition at various phenylglyoxal concentrations shows pseudo-first-order kinetics, with an apparent second-order rate constant of 14.2 M-1.min-1 for inactivation. The protective ability of several substrates and substrate analogues was investigated in order to ascertain if the inhibition was directed towards the active site of the enzyme. NADH and NAD+ did not protect but acyl carrier protein (ACP) and reduced coenzyme A, along with various derivatives, did protect. 9 microM ACP gave 35% protection from inactivation and 10 mM reduced coenzyme A gave 98% protection. The effectiveness of various subfragments of coenzyme A in protecting against inhibition indicates that the phosphate group is essential for preventing the binding of phenylglyoxal. The idea that phenylglyoxal is inhibiting by binding at the active site is further supported by the observation that the incorporation of 14C-labelled phenylglyoxal is directly related to the loss of activity. Extrapolation of the amount of label incorporated to give total inhibition shows that 4 mol of phenylglyoxal would be incorporated per mol of enzyme. This corresponds to the modification of two arginine side-chains with equal reactiveness towards the reagent. These results are consistent with there being two arginine residues either at the active site of the enzyme or in an environment which is protected from phenylglyoxal by a conformational change induced by coenzyme A binding.
Assuntos
Aldeídos/farmacologia , Brassica/enzimologia , Oxirredutases/antagonistas & inibidores , Fenilglioxal/farmacologia , Arginina/metabolismo , Sítios de Ligação , Cisteína/metabolismo , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Ativação Enzimática/efeitos dos fármacos , Cinética , Oxirredutases/metabolismo , Fenilglioxal/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
An antibody has been raised against rape seed enoyl-ACP reductase. This recognizes both the alpha and beta polypeptides of the enzyme. Immunoblotting of fresh seed demonstrates that beta is not present in seed material, and that it is produced by proteolysis during isolation. It is thus deduced that rape seed enoyl reductase is an alpha 4 homotetramer. Leaf material from both rape and Arabidopsis have an enoyl reductase with a similar electrophoretic mobility to the rape seed enzyme when analyzed on SDS-PAGE. Quantitative immunoassay has demonstrated that the enzyme continually increases during lipid deposition, indicating that an increase in this enzyme is required to sustain high levels of lipid biosynthesis. In vitro translation experiments show that the enzyme is nuclear coded and synthesized as a precursor form. Immunogold electron microscopy has demonstrated that enoyl reductase is located in plastids. It is shown that ACP-Sepharose may be used as a matrix in the purification of enoyl-ACP reductase.
Assuntos
Proteína de Transporte de Acila/metabolismo , Brassica/enzimologia , Oxirredutases/análise , Biossíntese de Proteínas , RNA Mensageiro/genética , Especificidade de Anticorpos , Western Blotting , Brassica/genética , Cromatografia de Afinidade , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Indução Enzimática , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Substâncias Macromoleculares , NAD/metabolismo , Oxirredutases/biossíntese , Oxirredutases/genética , Radioimunoensaio , Sementes/enzimologia , Especificidade por SubstratoRESUMO
3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
Assuntos
Oxirredutases do Álcool/química , Brassica/enzimologia , Ácido Graxo Sintases/química , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Catálise , Eletroforese em Gel de Poliacrilamida , Ácido Graxo Sintases/metabolismo , Imuno-Histoquímica , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Mapeamento de Peptídeos , Alinhamento de SequênciaRESUMO
The tetrameric, NADH-dependent enoyl acyl carrier protein reductase from developing seeds of Brassica napus (oil seed rape) has been crystallized from solutions containing ammonium sulphate as the precipitant in the presence of NAD+ or NADH using the hanging drop method of vapour diffusion. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with cell dimensions a = b = 70.5 A, c = 117.8 A. Considerations of the possible values of Vm indicate that the asymmetric unit contains a single subunit. The crystals are resistant to radiation damage and X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 1.9 A resolution. Determination of the structure of this enzyme will advance the understanding of the mechanisms of lipid biosynthesis in plants and provide an opportunity to study the interactions between this enzyme and its acyl carrier protein substrate.
Assuntos
Brassica/química , Oxirredutases/química , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)RESUMO
Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.
Assuntos
Escherichia coli/enzimologia , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , NAD/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Brassica/enzimologia , Sequência Conservada , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Ácidos Graxos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , NAD/química , Nucleotídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.
Assuntos
Compostos de Boro/química , Compostos de Boro/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Sítios de Ligação , Compostos de Boro/metabolismo , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , NAD/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Relação Estrutura-Atividade , Triclosan/química , Triclosan/metabolismo , Triclosan/farmacologiaRESUMO
Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.
Assuntos
Anti-Infecciosos Locais/química , Anti-Infecciosos Locais/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Triclosan/química , Triclosan/metabolismo , Sítios de Ligação , Compostos de Boro/metabolismo , Brassica/química , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Proteínas de Plantas/química , Conformação ProteicaRESUMO
Potato (Solanum tuberosum) tuber lectin is a chitin-binding, hydroxyproline-rich glycoprotein, which may be involved in the defence mechanism of the plant. We had previously obtained evidence that it consists of at least two very dissimilar domains. The aim was to use a combination of accurate determinations of molecular weight and protein sequencing to gain more accurate information on the domains. Accurate determinations of the molecular weight of the lectin by a MALDI mass spectrometer have shown that the subunit molecular weight is 65,500 (+/- 1100) and that of a totally deglycosylated sample is 31,250 (+/- 30). This means that the lectin is 52.3 (+/- 1)% carbohydrate with a considerable number of glycoforms being present. Partial sequences and other analyses are consistent with the existence of three distinct domains. These are: (1) an N-terminal region which is rich in proline but poor in hydroxyproline; (2) a glycosylated region with a glycosylated molecular weight of 45,300 (+/- 1100) and a deglycosylated molecular weight of 11,050 (+/- 50) which is extremely rich in glycosylated hydroxyproline residues with a similar sequence to extensins; and (3) a cystine-rich domain which has the sugar binding site shows partial conservation of a repeated motif common to many chitin-binding proteins of the hevin family including wheat-germ agglutinin. The closest similarity seems to be to the sequence of potato basic chitinase.
Assuntos
Lectinas/química , Lectinas/genética , Solanum tuberosum/química , Solanum tuberosum/genética , Aglutininas do Germe de Trigo/genética , Sequência de Aminoácidos , Sítios de Ligação , Quitina/metabolismo , Sequência Conservada , Cistina/química , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hidroxiprolina/química , Lectinas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Lectinas de Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Solanum tuberosum/metabolismoRESUMO
The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT.
Assuntos
Aciltransferases/genética , Brassica/genética , DNA Complementar/genética , Genes de Plantas , Proteína de Transporte de Acila S-Maloniltransferase , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/análise , Escherichia coli/genética , Proteínas de Escherichia coli , Ácido Graxo Sintase Tipo II , Dados de Sequência Molecular , Mutação , Homologia de Sequência de AminoácidosRESUMO
In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3-oxoacyl-ACP reductase. Using a novel radio-chemical assay for 3-oxoacyl-ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post-emergence, but slowed after this point even though 3-oxoacyl-ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.
Assuntos
Oxirredutases do Álcool/metabolismo , Brassica/crescimento & desenvolvimento , Brassica/metabolismo , Ácidos Graxos/biossíntese , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase , Brassica/citologia , Brassica/enzimologia , Extratos Celulares , Ácidos Graxos/análise , Cinética , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Espectrofotometria , TrítioRESUMO
Enoyl-ACP reductase, a component of fatty acid synthase, is a target for anti-microbial agents and herbicides. Here we demonstrate the kinetic mechanism to be a compulsory-order ternary complex with NADH binding before the acyl substrate. Matrix-assisted laser desorption ionisation mass spectrometry analysis of enzymatically and synthesised crotonyl-ACP substrate showed the former to contain a single acyl group, whereas the latter contained up to four additional crotonylations. The use of authentic crotonyl-ACP will be important in future kinetic and crystallographic studies.
Assuntos
Brassica/enzimologia , Oxirredutases/metabolismo , Ligação Competitiva , Catálise , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Estabilidade Enzimática , Cinética , Peso Molecular , Oxirredutases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Sequence analysis of several cDNAs encoding the phasin protein of Ralstonia eutropha indicated that the carboxyl terminus of the resulting derived protein sequence is different from that reported previously. This was confirmed by: (1) sequencing of the genomic DNA; (2) SDS-PAGE and peptide analysis of wild-type and recombinant phasin; and (3) mass spectrometry of wild-type phasin protein. The results have implications for the model proposed for the binding of this protein to polyhydroxyalkanoic acid granules in the bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , Hidroxiácidos/metabolismo , Lectinas/química , Lectinas/metabolismo , Lectinas de Plantas , Alcaligenes , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , DNA Complementar/genética , Lectinas/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Análise de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The sequence of the 3' 1462nts of RNA-1 of a UK isolate of the fungal-transmitted virus barley mild mosaic (BaMMV) has been determined. An open reading frame encoding the coat protein gene was identified within this region using amino acid sequence information obtained by cyanogen bromide cleavage of virus particles. The amino acid sequence of the full-length coat protein was deduced from the nucleotide sequence. Amino acid sequence comparisons revealed highest homology to the coat protein of barley yellow mosaic virus. In addition, a significant, but limited, number of the amino acid residues that are conserved between aphid-transmitted potyviruses were also conserved between BaMMV and potyviruses.
Assuntos
Vírus do Mosaico/genética , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/genética , Clonagem Molecular , Genes Virais , Hordeum/microbiologia , Dados de Sequência Molecular , Vírus do Mosaico/classificação , RNA Viral/genética , Análise de Sequência de RNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single-chain Fv (scFv), displayed on filamentous phage. Analysis of the selected clones by BstNI fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes. The binding specificities were confirmed by Western blots and ELISA. The scFv preparations reacted with B. napus ENR, but not with beta-keto reductase, nor enoyl reductase from Escherichia coli. Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognized an epitope located within the N-terminal 80 amino acids of the enzyme molecule. The scFv were used to detect ENR directly in extracts of B. napus seeds.